Methods for treating microglial dysfunction

ABSTRACT

Among the various aspects of the present disclosure is the provision of methods of treating a microglial dysfunction-associated diseases, disorder, and conditions. The present disclosure provides for a method for treating microglial dysfunction in a subject having a microglial dysfunction-associated neurodegenerative disease comprising administering to a subject a therapeutically effective amount of a microglial rescuing agent. The present disclosure also provides for a method of reversing neuronal damage in a subject having a microglial dysfunction-associated neurodegenerative disease, wherein the microglial dysfunction-associated neurodegenerative disease is characterized by a single nucleotide polymorphisms (SNPs) or mutation in Trem2 or ApoE affecting microglial functions.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.62/529,753, filed Jul. 7, 2017, the disclosure of which is herebyincorporated by reference in its entirety.

GOVERNMENTAL RIGHTS

This invention was made with government support under grant numbersAG051485, AG005681, and CA009547 awarded by National Institutes ofHealth. The government has certain rights in the invention.

FIELD OF THE INVENTION

The present disclosure generally relates to compositions and methods oftreating a microglial dysfunction associated disease, disorder, orcondition.

BACKGROUND

Alzheimer's disease (AD) is the most common cause of late onsetdementia. AD lesions in the CNS include plaques of amyloid β (Aβ)peptides and neurofibrillary tangles of hyperphosphorylated tau protein,both linked to synapse loss, neuronal death, and ultimately cognitivedecline (Holtzman et al., 2011; Huang and Mucke, 2012). Rare familial ADis due to mutations in amyloid precursor protein (APP) and presenilins(PS) that promote the generation of Aβ peptides prone to aggregation(Tanzi, 2012). However, the risk for common late-onset AD is associatedwith rare variants of immune receptors expressed on microglia (Guerreiroand Hardy, 2014; Tanzi, 2012). One of these receptors, TREM2, recognizesphospholipids, apoptotic cells, and lipoproteins (Atagi et al., 2015;Bailey et al., 2015; Wang et al. 2015; Yeh et al., 2016). TREM2transmits intracellular signals through two adapters, DAP12 and DAP10,which recruit protein tyrosine kinase Syk and phosphatidylinositol3-kinase (PI3-K), respectively (Peng et al., 2010).Arginine-to-histidine variants at position 47 (R47H) or 62 (R62H) ofTREM2 increase the risk for sporadic AD and impair binding tophospholipid ligands (Atagi et al., 2015; Bailey et al., 2015; Guerreiroand Hardy, 2014; Wang et al., 2015; Yeh et al., 2016). These variants,as well as TREM2 deficiency and haploinsufficiency in mouse models ofAD, moderate microglial proliferation, survival, and accumulation aroundAβ plaques, thereby facilitate Aβ plaque buildup and injury of adjacentneurons (Jay et al., 2017; Ulrich et al., 2014; Wang et al., 2015, 2016;Yuan et al., 2016). TREM2 has also been implicated in microglialphagocytosis of dead neurons, damaged myelin, and Aβ plaques (Neumannand Takahashi, 2007; Yeh et al., 2016). However, why defective TREM2function or expression affects microglia responses to AD lesions is notknown.

SUMMARY

Among the various aspects of the present disclosure is the provision ofmethods of treating a microglial dysfunction-associated diseases,disorder, and conditions.

One aspect of the present disclosure is directed to a method of treatingmicroglial dysfunction in a subject having a microglialdysfunction-associated neurodegenerative disease comprisingadministering to a subject a therapeutically effective amount of amicroglial rescuing agent. In some embodiments, the microglial rescuingagent comprises creatine, a creatine analog, or pharmaceuticallyacceptable salt thereof.

Another aspect of the present disclosure is directed to a method oftreating microglial dysfunction in a subject having a microglialdysfunction-associated neurodegenerative disease comprisingadministering to a subject a therapeutically effective amount ofdectin-1 ligand.

An additional aspect of the present disclosure is directed to a methodof suppressing microglial autophagy in a subject having a microglialdysfunction-associated neurodegenerative disease.

In yet another aspect of the present disclosure is directed to a methodof reversing neuronal damage in a subject having a microglialdysfunction-associated neurodegenerative disease, wherein the microglialdysfunction-associated neurodegenerative disease is characterized by asingle nucleotide polymorphisms (SNPs) or mutation in Trem2 or ApoEaffecting microglial functions.

BRIEF DESCRIPTION OF THE FIGURES

The application file contains at least one drawing executed in color.Copies of this patent application publication with color drawing(s) willbe provided by the Office upon request and payment of the necessary fee.

FIG. 1A, FIG. 1B, FIG. 1C, FIG. 1D, FIG. 1E and FIG. 1F depict a defectin TREM2 enhances autophagy in vivo in the 5XFAD mouse model and in ADpatients. FIG. 1A CD45⁺, CD11b⁺, F4/80⁺ cells were sorted from mousebrains of WT, Trem2^(−/−), 5XFAD, and Trem2^(−/−) 5XFAD mice. TEM imagesof microglia sorted from 8-month-old WT, Trem2^(−/−), 5XFAD, andTrem2^(−/−), 5XFAD mice. FIG. 1B average number of multivesicular andmultilamellar structures/cell (30 cells analyzed/genotype). FIG. 1Cconfocal images of plaque bearing regions of the cortex (1.1 mm Bregmato 0.8 mm Bregma) of 8-month-old WT, Trem2^(−/−), 5XFAD, and Trem2^(−/−)5XFAD mice show Iba-1⁺ microglia (red), methoxy X04⁺ plaques (blue), andLC3 (green). Z-stacks composed of ˜30 images taken at 1.2 μm intervalswere analyzed. Results are reported as an average of 2 regions ofinterest (ROI) analyzed. FIG. 1D quantification of the % of microgliathat are positive for LC3 puncta. ˜150-400 microglia/HPF were analyzeddepending on the genotype of the animal. FIG. 1E confocal images ofsections from post-mortem brains of R47H⁺ AD patients and case-matchedcontrols (CV, common variant of TREM2) show Iba-1⁺ microglia (red),methoxy X04⁺ plaques (blue), and LC3 (green). 3 ROIs/donor were analyzedand a total of between 400 and 700 microglia/individual were analyzed.FIG. 1F percentages of LC3+ microglia in post mortem specimens of ADpatients with different genotypes. ***p<0.005, ****p<0.001 by One-wayANOVA with Holm-Sidak's multiple comparisons test. 15 cells from 2separate mice were visualized for TEM (FIG. 1A, FIG. 1B). Confocalimages are representative of 3 female mice per group (FIG. 1C) or 7R47H, 4 R62H, and 8 case matched AD patients for post-mortem specimens(FIG. 1E). Immunoblots are representative of 3 independent experimentsfrom microglia from 3 separate mice per group (FIG. 1G). Arrowheadsindicate multilamellar and multivesicular structures (FIG. 1A) or LC3+vesicles (FIG. 1C, FIG. 1E). See also FIG. 8 and Table 1.

FIG. 2A, FIG. 2B, FIG. 2C, FIG. 2D, FIG. 2E and FIG. 2F depict a defectin TREM2 impairs mTOR activation and elicits AMPK activation, autophagyand cell death in microglia from 5XFAD mice. FIG. 2A microglia weresorted as in FIG. 1A. Immunoblots for LC3I/II, p62, phosphoserine 473AKT, phospho-AMPK, phospho-NDRG1, phospho-4EBP1, phospho-757 ULK1, andβ-actin were performed on cell lysates. FIG. 2B quantification of theLC3II/I ratio in microglia from different genotypes. FIG. 2C single cellsuspensions of brain tissue were incubated with MitoTraker Green andstained for CD45⁺, CD11b⁺, F4/80⁺. Representative histograms comparingunstained cells and microglia from 5XFAD and Trem2^(−/−) 5XFAD mice areshown. FIG. 2D quantification of the geometric mean fluorescenceintensity (gMFI) of microglia from 3 mice of each genotype is shown.FIG. 2E confocal images of brain sections of 8 month-old WT,Trem2^(−/−), 5XFAD, and Trem2^(−/−) 5XFAD mice were taken as in FIG. 1C.Images depict Iba-1⁺ microglia (red), methoxy X04⁺ plaques (blue), andcleaved caspase-3 (green). FIG. 2F quantification of the % of LC3⁺microglia that are positive for cleaved caspase-3. ****p<0.001 byOne-way ANOVA with Holm-Sidak's multiple comparisons test (FIG. 2B andFIG. 2F). ** p<0.01 by Student's T test (FIG. 2D). Immunoblots arerepresentative of 3 independent experiments from microglia from 3separate mice per group (FIG. 2A). Confocal images are representative of3 female mice per group (FIG. 2E). See also FIG. 9.

FIG. 3A, FIG. 3B, FIG. 3C, FIG. 3D, FIG. 3E, FIG. 3F, FIG. 3G, FIG. 3H,FIG. 3I, FIG. 3J and FIG. 3K depict TREM2 deficiency affects mTORsignaling and induces autophagy in BMDM. FIG. 3A TEM images of WT andTrem2^(−/−) BMDM cultured overnight in either in 10% or 0.5% LCCM assource of CSF1. FIG. 3B number of multivesicular structures/cellobserved in the TEM images 30 cells/genotype and condition wereanalyzed. FIG. 3C quantification of the LC3II/LC3I ratio in BMDMs fromWT and Trem2^(−/−) mice cultured in 10% or 0.5% LCCM overnight orstarved in HBSS for 4 hours prior to lysis. FIG. 3D immunoblots for LC3and actin performed on lysates from WT and Trem2^(−/−) BMDMs cultured in10% or 0.5% LCCM overnight. Cell were treated with bafilomycin weretreated for 5 hours prior to harvest at a final concentration of 0.5μg/ml. FIG. 3E quantification of LC3II/LC3I ratio in BMDMs from WT andTrem2^(−/−) mice treated as indicated. FIG. 3F-FIG. 3H immunoblots forphosphorylated Akt, NDRG1, S6K, 4EBP1, AMPK, Ulk1 and relative controls.Lysates were from WT and Trem2^(−/−) BMDM cultured overnight in 10% or0.5% LCCM. FIG. 3I immunoblots for phosphorylated Akt, NDRG1, S6K,4EBP1, mTOR, total S6K, Akt, and actin performed on lysates from WT andTrem2^(−/−) BMDMs cultured overnight in 10% or 0.5% LCCM followed by theaddition of wortmannin for 3 hours prior to harvest. FIG. 3J and FIG. 3Kimmunoblots for LC3 and phosphoserine 473 AKT in WT and Trem2^(−/−) BMDMcultured in 10% LCCM with the indicated concentration of tunicamycin.Bar graph shows LC3II/LC3I ratios. Error bar represents mean±SEM.*p<0.05, **p<0.01, or ****p<0.001 by One-way ANOVA with Holm-Sidak'smultiple comparisons test (FIG. 3B, FIG. 3C, FIG. 3E, FIG. 3K). Data arerepresentative of at least 3 independent experiments. Arrowheadsindicate multilamellar and multivesicular structures (FIG. 3A). See alsoFIG. 10.

FIG. 4A, FIG. 4B, FIG. 4C, FIG. 4D, FIG. 4E, FIG. 4F and FIG. 4G depictTREM2 deficiency reduces anabolic and energetic metabolism in BMDM. FIG.4A top most changed metabolites between WT and Trem2^(−/−) BMDM culturedovernight in 10% LCCM. Defined as p≤0.01 and identified in the mousemetabolic network analysis in B. FIG. 4B shiny-genes and metabolites(GAM) output for network analysis combining mass spectrometry andRNA-seq data highlights differences between WT and Trem2^(−/−) BMDMcultured in 10% LCCM. Enzyme-encoding mRNAs and metabolitesdownregulated or upregulated in Trem2^(−/−) cells vs WT cells areindicated with green or red nodes and connecters, respectively. FIG. 4Ctop most changed metabolites between WT and Trem2^(−/−) BMDM cultured in0.5% LCCM. Defined as p≤0.01 and identified in the mouse metabolicnetwork analysis in FIG. 11C. FIG. 4D ATP content of WT and Trem2^(−/−)BMDM cultured in the indicated concentration of LCCM overnight. FIG. 4Eextracellular acidification rate (ECAR) and baseline oxygen consumptionrate (OCR) by WT and Trem2^(−/−) BMDM cultured overnight in theindicated concentration of LCCM. FIG. 4F and FIG. 4G mitochondrial massof WT and Trem2^(−/−) BMDM assessed by Mito Tracker Green incorporationand by the ratio of mitochondrial-to nuclear DNA. Error bar representsmean±SEM. *p<0.05, ** p<0.01, or ****p<0.001 by One-way ANOVA withHolm-Sidak's multiple comparisons test (FIG. 4C) or Student's T test(FIG. 4F, FIG. 4G). Data are representative of at least 3 independentexperiments. See also FIG. 11.

FIG. 5A, FIG. 5B, FIG. 5C, FIG. 5D, FIG. 5E, FIG. 5F and FIG. 5G depictenhanced energy storage or dectin-1 signaling can compensate for TREM2deficiency. FIG. 5A ECAR of WT and Trem2^(−/−) BMDM incubated overnightin 0.5% LCCM±10 mM cyclocreatine. FIG. 5B viability of WT andTrem2^(−/−) BMDM incubated for 40 hours in 0.5% LCCM±cyclocreatine. FIG.5C Immunoblots of LC3, phosphorylated mTOR, phosphorylated Akt, andactin in WT and Trem2^(−/−) BMDM incubated overnight in 0.5% LCCM±5 mMcyclocreatine. FIG. 5D and FIG. 5F LC3, phosphoserine 473 AKT, p62, andactin immunoblots from WT and Trem2^(−/−) BMDM incubated overnight inthe indicated concentration of LCCM±depleted zymosan. FIG. 5EQuantification of the LC3II/LC3I ratio derived from immunoblots of LC3as shown in D. FIG. 5G ATP content of WT and Trem2^(−/−) BMDM culturedin the indicated concentration of LCCM±zymosan overnight. *p<0.05 or****p<0.001 by One-way ANOVA with Holm-Sidak's multiple comparisons test(FIG. 5B, FIG. 5E, FIG. 5G). Data are representative of results from atleast 3 independent experiments.

FIG. 6A, FIG. 6B, FIG. 6C, FIG. 6D, FIG. 6E and FIG. 6F depict enhancedenergy storage can compensate for TREM2 deficiency in vivo. FIG. 6A TEMimages of microglia sorted from 8-month-old 5XFAD, and Trem2^(−/−) 5XFADmice±cyclocreatine. FIG. 6B quantification of the number ofmultivesicular and multilamellar structures/cell from A. FIG. 6Cconfocal images of brain sections of 8-month-old 5XFAD, and Trem2^(−/−)5XFAD mice±cyclocreatine show Iba-1⁺ microglia (red), methoxy X04⁺plaques (blue), and LC3 (green). FIG. 6D clustering analysis quantifyingthe number of microglia per mm³ within 15 μm of the surface of plaques.FIG. 6E quantification of the number of LC3 puncta per HPF in thecortexes of the indicated mice. FIG. 6F quantification of the percentageof microglia that were cleaved caspase-3 positive from the indicatedmice. *p<0.05, ***p<0.005 and ****p<0.001 by One-way ANOVA withHolm-Sidak's multiple comparisons test (FIG. 6B, FIG. 6D-FIG. 6F)results pooled from 2 independent experiments representing a total of5-8 male and female mice per treatment group. Arrowheads indicatemultilamellar and multivesicular structures (FIG. 6A) or LC3⁺ vesicles(FIG. 6C). See also FIG. 12.

FIG. 7A, FIG. 7B, FIG. 7C, FIG. 7D, FIG. 7E, FIG. 7F and FIG. 7G depictEnergy supplementation can compensate for TREM2 deficiency and decreaseneuronal damage in vivo. FIG. 7A representative images depicting plaques(X04 in blue), nuclei (To-Pro3 in white), microglia (Iba-1 in red), andSpp1 (in green) staining in cortexes of mice from the indicatedgenotypes. FIG. 7B quantification of the percentage of microglia thatwere Spp1⁺ in the indicated genotypes of mice. Confocal images weretaken as in FIG. 1C. FIG. 7C immunoblots performed on lysates ofmicroglia sorted from the indicated genotype and treatment group ofmice. Immunoblots for phosphorylated Akt, NDRG1, total LC3, Akt, andactin. FIG. 7D quantification of the LC3II/LC3I ratio observed inimmunoblots from 3 mice of each of the indicated genotypes and treatmentgroups. FIG. 7E average intensity of the plaques observed in thecortexes of mice from the indicated genotypes and treatment groups. FIG.7F representative images depicting plaques (X04 in blue), nuclei(To-Pro3 in white), and N-terminus APP (green) from the indicated miceand treatment groups. Confocal images were taken as in FIG. 1C. FIG. 7Gquantification of the number of dystrophic neurites/plaque in theindicated mice and treatment group. N.S. indicates not significant,*p<0.05, and ****p<0.001 by One-way ANOVA with Holm-Sidak's multiplecomparisons test (FIG. 7A, FIG. 7C, FIG. 7D, FIG. 7F) results pooledfrom 2 independent experiments representing a total of 5-8 male andfemale mice per treatment group. See also FIG. 12.

FIG. 8 depicts a wider field of view of LC3 in microglia. Related toFIG. 1. Lower magnification confocal images of cortexes of WT,Trem2^(−/−), 5XFAD and Trem2^(−/−)5XFAD mice. Confocal images arerepresentative of 3 female mice per group.

FIG. 9A, FIG. 9B, FIG. 9C, FIG. 9D, FIG. 9E, FIG. 9F, FIG. 9G, FIG. 9H,and FIG. 9I depict TREM2 deficiency in an AD model affects microgliaexpression of genes involved in metabolic pathways and microglia fromTrem2^(−/−) 5XFAD mice undergo more cell death. Related to FIG. 2. FIG.9A Sqstm1 expression taken from microarrays of sorted microglia from 8month old WT, Trem2^(−/−), 5XFAD and Trem2^(−/−) 5XFAD mice. FIG. 9B top10 pathways in IPA analysis of differentially expressed genes from 5XFADand Trem2^(−/−) 5XFAD microglia. Negative log₁₀ p-values are shown. FIG.9C gene enrichment plots for genes included in the eIF2, glycolysis, andmTOR signaling modules of IPA. Plots were generated utilizing gene-setenrichment analysis (GSEA) software. FIG. 9D-FIG. 9F heat maps comparingWT, 5XFAD, Trem2^(−/−), and Trem2^(−/−) 5XFAD microglia for expressionof genes included in the eIF2, glycolysis and mTOR signaling pathways.FIG. 9G illustration of the glycolytic pathway: proteins indicated inred correspond to genes upregulated in 5XFAD but not Trem2^(−/−) 5XFADmicroglia compared to WT microglia. FIG. 9H mosaic of images depictingrepresentative images of microglia (Iba1 red), plaques (methoxy-X04blue), cleaved caspase-3 (aqua), LC3 (green), and total merged imagesfrom a 5XFAD and a Trem2^(−/−) 5XFAD animal. FIG. 9I quantification ofthe percentage of microglia that are both LC3 and cleaved caspase-3positive. Microarray data represents analyses of microglia sorted from 3WT, 4 Trem2^(−/−), 5 5XFAD, and 5 Trem2^(−/−) 5XFAD mice (FIG. 9A-FIG.9G). Confocal images are representative of 3 female mice per group (FIG.9H). ***p<0.005 by One-way ANOVA with Holm-Sidak's multiple comparisonstest (FIG. 9I).

FIG. 10A and FIG. 10B depict Trem2 is activated in vitro and contributesto PI3K-dependent mTOR activation. Related to FIG. 3. FIG. 10Aimmunoblots for phosphorylated Akt, NDRG1, S6K, 4EBP1, mTOR, total S6K,Akt, and actin performed on lysates from WT and Trem2^(−/−) BMDMscultured overnight in 10% or 0.5% LCCM followed by the addition ofLy294002 for 3 hours prior to harvest. FIG. 10B reporter cell assayassessing TREM2 activation in reporter cell line incubated at optimaland low serum conditions with or with soluble anti-TREM2. N.S. indicatesnot significant and ****p<0.001 by One-way ANOVA with Holm-Sidak'smultiple comparisons test (FIG. 10B). Results are representative of atleast 3 independent experiments.

FIG. 11A, FIG. 11B, FIG. 11C, FIG. 11D, FIG. 11E, FIG. 11F, FIG. 11G,FIG. 11H, FIG. 11I, FIG. 11J, FIG. 11K, FIG. 11L and FIG. 11M showmacrophages and microglia from Trem2^(−/−) mice are less energeticallyactive. Related to FIG. 4. FIG. 11A, FIG. 11B heatmaps representing thetotal metabolic profiles of WT compared to Trem2^(−/−) BMDMs cultureovernight in 10% LCCM (A) or 0.5% LCCM (FIG. 11B). FIG. 11C Shiny-GAMoutput for network analysis combining RNA-seq and mass spectrometry datahighlights differences between WT and Trem2^(−/−) BMDM culturedovernight in 0.5% LCCM. Enzymes-encoding mRNAs and metabolites that aredownregulated or upregulated in Trem2^(−/−) cells vs. WT cells arerepresented by green or red nodes and connecters, respectively. FIG. 11Dquantification of phosphocreatine from WT and Trem2^(−/−) BMDMs culturedin the indicated concentration of LCCM. FIG. 11E, FIG. 11F assessmentand quantification of the mitochondrial mass of resident peritonealmacrophages from WT and Trem2^(−/−) mice by MitoTraker Greenincorporation. FIG. 11G, FIG. 11H assessment and quantification of themitochondrial mass of thioglycolate elicited peritoneal macrophages fromWT and Trem2^(−/−) mice by MitoTracker Green incorporation. FIG. 11I ATPcontent of WT and Trem2^(−/−) microglia cultured in 10% LCCM overnight.FIG. 11J mitochondrial content of WT and Trem2^(−/−) microglia assessedby the ratio of mitochondrial-to nuclear DNA. FIG. 11K extracellularacidification rate (ECAR) of WT and Trem2^(−/−) microglia culturedovernight in 10% LCCM. FIG. 11L primary WT and Trem2^(−/−) microgliawere incubated overnight in the indicated concentration of LCCM.Immunoblots for LC3, p757 Ulk1, p317 Ulk1, p473 Akt, pNDRG1, and β actinwere performed. FIG. 11M quantification of the LC3II/LC3I ratio fromimmunoblot performed on primary microglia shown in FIG. 11L. *p<0.05 or****p<0.001 by One-way ANOVA with Holm-Sidak's multiple comparisons test(FIG. 11 FIG. 11, FIG. 11F, FIG. 11I, FIG. 11H, FIG. 11K). Results arerepresentative of at least 3 independent experiments (FIG. 11E-FIG. 11K)or 2 independent experiments (FIG. 11L, FIG. 11M).

FIG. 12A, FIG. 12B, FIG. 12C, FIG. 12D, FIG. 12E, FIG. 12F, FIG. 12G,FIG. 12H, and FIG. 12I depict enhanced energy storage improvesmicroglial response in Trem2^(−/−) 5XFAD mice in vivo. Related to FIG. 6and FIG. 7. FIG. 12A quantification of the number of microglia perhigh-powered field in the cortexes of brains from mice of the indicatedgenotype and treatment group. FIG. 12B quantification of the percentageof microglia that contain LC3 puncta in the cortexes of brains from miceof the indicated genotype and treatment group. FIG. 12C quantificationof the number of LC3 puncta per LC3⁺ microglia. FIG. 12D quantificationof the relative enrichment of LC3⁺ microglia within 15 μm of a plaquesurface in the cortexes of brains from mice of the indicated genotypeand treatment group. FIG. 12E quantification of the relative enrichmentof cleaved caspase-3⁺ microglia within 15 μm of a plaque surface in thecortexes of brains from mice of the indicated genotype and treatmentgroup. FIG. 12F and FIG. 12G the percent of the overall area of thecortex (FIG. 12F) and hippocampus (FIG. 12G) 1.5× brighter than the meanin methoxy-X04 stained sections from mice of the indicated genotype andtreatment group. FIG. 12H quantification of the percentage of microgliacontaining methoxy-X04 in plaque bearing regions of the cortexes frommice of the indicated genotype and treatment group. FIG. 12I assessmentof the complexity of plaques in the cortexes from mice of the indicatedgenotype and treatment group. N.S. indicates not significant, **p<0.01or ****p<0.001 by One-way ANOVA with Holm-Sidak's multiple comparisonstest (FIG. 12A, FIG. 12B, FIG. 12D, FIG. 12E). Results pooled from 2independent experiments representing a total of 5-8 male and female miceper treatment group.

DETAILED DESCRIPTION

Applicants have discovered that the use of a microglial rescuing agentwhich supplements microglial energetic metabolism can be an effectivetreatment for subjects with microglial dysfunction-associated diseases,disorder, and conditions.

Disclosed herein are components used to prepare disclosed compositionsas well as the compositions themselves, and methods of use thereof. Itis understood that when combinations, subsets, interactions, groups,etc. of these materials are disclosed, that while specific reference toeach various individual and collective combinations and permutation maynot be explicitly disclosed, each is specifically contemplated anddescribed herein. For example, if a particular compound is disclosed anddiscussed and a number of modifications that can be made to a number ofmolecules of the compound are discussed, specifically contemplated iseach and every combination and permutation of the compound and themodifications that are possible unless specifically indicated to thecontrary. Thus, if a class of molecules A, B, and C are disclosed aswell as a class of molecules D, E, and F and an example of a combinationmolecule, A-D is disclosed, then even if each is not individuallyrecited each is individually and collectively contemplated, meaningcombinations, A-E, A-F, B-D, B-E, B-F, C-D, C-E, and C-F are considereddisclosed. Likewise, any subset or combination of these is alsodisclosed. Thus, for example, the sub-group of A-E, B-F, and C-E wouldbe considered disclosed. This concept applies to all aspects of thisapplication including, but not limited to, steps in methods of makingand using the disclosed compositions. Thus, if there are a variety ofadditional steps that can be performed it is understood that each ofthese additional steps can be performed with any specific embodiment orcombination of embodiments of the disclosed methods.

Additional aspects of the invention are described below.

(I) Compositions

One aspect of the present disclosure encompasses a microglial rescuingagent capable of mitigating one or more of the pathologies associatedwith microglial dysfunction. In some embodiments microglial dysfunctionresults from perturbations of cellular biosynthetic metabolism. In oneaspect, microglial dysfunction results from impaired mTOR activation. Insome embodiments, the present disclosure encompasses providing atherapeutically effective amount of one or more microglial rescuingagents, which results in improved metabolic activity, decreasedautophagy, decreased cell death, improved microglia viability, improvedmicroglia numbers or a combination thereof.

A composition of the invention may optionally comprise one or moreadditional drugs or therapeutically active agents in addition to themicroglial rescuing agent. A composition of the invention may furthercomprise a pharmaceutically acceptable excipient, carrier, or diluent.Further, a composition of the invention may contain preserving agents,solubilizing agents, stabilizing agents, wetting agents, emulsifiers,sweeteners, colorants, odorants, salts (substances of the presentinvention may themselves be provided in the form of a pharmaceuticallyacceptable salt), buffers, coating agents, or antioxidants.

The compositions as disclosed herein comprise microglial rescuing agentssuch as creatine compounds, a creatine analogs, and pharmaceuticallyacceptable salts thereof. Additionally, the compositions as disclosedherein comprise activators of the dectin-1 pathway, such as dectin-1ligands. The present disclosure also provides pharmaceuticalcompositions. The pharmaceutical composition comprises a microglialrescuing agent, as an active ingredient, and at least onepharmaceutically acceptable excipient.

Other aspects of the invention are described in further detail below.

(a) Creatine Compounds

Creatine compounds useful in the present invention include compoundswhich modulate microglial metabolism. As described herein, creatine andderivatives/analogs thereof have been identified to rescue microglialfunction and treat microglial-dysfunction associated disease. Creatineand analogs thereof have been shown to supplement microglial energeticmetabolism in subjects with microglial dysfunction, particularly thosewith a TREM2 or ApoE variant. Compounds which are effective for thispurpose include creatine, creatine phosphate and analogs thereof,compounds which mimic their activity, and salts of these compounds asdefined herein. Exemplary creatine compounds are described below.

Creatine (also known as N-(aminoiminomethyl)-N-methylglycine;methylglycosamine or N-methyl-guanido acetic acid) is a known substance.(See, The Merck Index, 60 Eleventh Edition, No. 2570 (1989).

Creatine is phosphorylated chemically or enzymatically by creatinekinase to generate creatine phosphate, which also is known (see, TheMerck Index, No. 7315). Both creatine and creatine phosphate(phosphocreatine) can be extracted from animal tissue or synthesizedchemically. Both are commercially available.

Cyclocreatine is an essentially planar cyclic analog of creatine.Although cyclocreatine is structurally similar to creatine, the twocompounds are distinguishable both kinetically and thermodynamically.Cyclocreatine is phosphorylated efficiently by creatine kinase in theforward reaction both in vitro and in vivo. Rowley, G. L., J. Am. Chem.Soc. 93: 5542-5551 (1971); McLaughlin, A. C. et. al., J. Biol. Chem.247, 4382-4388 (1972).

The phosphorylated compound phosphocyclocreatine is structurally similarto phosphocreatine; however, the phosphorous-nitrogen (P N) bond ofcyclocreatine phosphate is more stable than that of phosphocreatine.LoPresti, P. and M. Cohn, Biochem. Biophys. Acta 998: 317-320 (1989);Annesley, T. M. and J. B. Walker, J. Biol. Chem. 253; 8120-8125, (1978);Annesley, T. M. and J. B. Walker, Biochem. Biophys. Res. Commun. 74:185-190 (1977).

A creatine analog can be any creatine analog that targets the creatinekinase system or a creatine based composition. For example, a creatineanalog can be cyclocreatine, phosphocreatine (aka creatine phosphate),nicotinamide mononucleotide (NMN), creatine ethyl ester, creatinenitrate, creatine gluconate, creation methyl ester, creatine riboside,creatine sulphate, serotonin creatine sulphate, creatine ethylester(HCl), creatine hydrochloride, creatine pyruvate, creatine citrate,creatine hemisulfate salt, creatine-(methyl-d3) monohydrate, creatinezinc chloride, creatine taurinate, 5,7-dihydroxytryptamine, L-argininealpha-ketoglutarate, creatine pyroglutamate, creatine calcium, creatinemagnesium, creation dextrose, creatine ethyl ester malate, orderivatives thereof. Exemplary compounds are shown below:

(b) Dectin-1 Agonist

Dectin-1 (aka C-type lectin domain family 7 member A; UniProt accessionnumber Q9BXN2) is a protein that in humans is encoded by the CLEC7Agene. Dectin-1 is a member of the C-type lectin/C-type lectin-likedomain (CTL/CTLD) superfamily. The encoded glycoprotein is a small typeII membrane receptor with an extracellular C-type lectin-like domainfold and a cytoplasmic domain with a partial immunoreceptortyrosine-based activation motif. It functions as a pattern-recognitionreceptor for a variety of β-1,3-linked and β-1,6-linked glucans fromfungi and plants, and in this way plays a role in innate immuneresponse. Expression is found on myeloid dendritic cells, monocytes,macrophages and B cells. Alternate transcriptional splice variants,encoding different isoforms, have been characterized. This gene isclosely linked to other CTL/CTLD superfamily members on chromosome 12p13in the natural killer gene complex region. Dectin-1 is a transmembraneprotein containing an immunoreceptor tyrosine-based activation(ITAM)-like motif in its intracellular tail (which is involved incellular activation) and one C-type lectin-like domain(carbohydrate-recognition domain, CRD) in the extracellular region(which recognizes β-glucans and endogenous ligands on T cells). The CRDis separated from the membrane by a stalk region. CLEC7A containsputative N-linked sites of glycosylation in the stalk region.

The C-type lectin receptors are class of signaling pattern recognitionreceptors which are involved in antifungal immunity, but also playimportant roles in immune responses to other pathogens such as bacteria,viruses and nematodes. As a member of this receptor family, Dectin-1recognizes β-glucans and carbohydrates found in fungal cell walls, somebacteria and plants, but may also recognize other unidentified molecules(endogenous ligand on T-cells and ligand on mycobacteria). Ligandbinding induces intracellular signaling via the ITAM-like motif. CLEC7Acan induce both Syk dependent or Syk independent pathways. Dimerizationof dectin-1 upon ligand binding leads to tyrosine phosphorylation by Srcfamily kinases and recruitment of Syk. Syk activates transcriptionfactor NFκB. This transcription factor is responsible for the productionof numerous inflammatory cytokines and chemokines such as TNF, IL-23,IL-6, IL-2. Other responses include: respiratory burst, production ofarachidonic acid metabolites, dendritic cell maturation, andphagocytosis of the ligand.

The term “Dectin-1 agonist”, also referred to herein as “Dectin-1ligand”, refers to a molecule that specifically binds to Dectin-1resulting in activation of the Dectin-1 pathway. Activators of Dectin-1signaling may be used alone, with other agents with similar or differenteffects or with other modalities, including surgery and the like.

In one aspect, this disclosure provides a microglial rescuing agentcapable of activating the Dectin-1 signaling pathway. A Dectin-1 agonistis also referred to herein as a ligand. In one embodiment, the Dectin-1agonistic is an antibody or a fragment thereof. In another embodiment,the Dectin-1 agonistic is a small molecule. Non-limiting examples ofDectin-1 ligands include beta-glucan peptide (BGP), curdlan AL,heat-killed C. albicans, heat-killed S. cerevisiae, laminarin, lichenan,pustulan, schizophyllan, scleroglucan, WGP Dispersible, Zymosan, ZymosanDepleted. These agonists are commercially available (Invivogen). Anotherexample of Dectin-1 activator is vimentin. Another example of Dectin-1activator is an agonistic anti-Dectin-1 antibody, such as, for example,an antibody described in U.S. Pat. No. 9,045,542, the description ofwhich antibody is incorporated herein by reference. In one embodiment, amicroglial rescuing agent is one or more of Dectin-1 agonists oractivators.

In one aspect, this disclosure provides methods for identifyingactivators of Dectin-1 pathway in microglial cells. In one embodiment,the activators of Dectin-1 pathway are Dectin-1 agonists. The activityof a test agent may be evaluated based on the effect on any step of theDectin-1 pathway (as described in this disclosure). It can be comparedto the effect in the absence of the test compound or may be compared tothe effect of Dectin-1 or a known agonist thereof.

Assays to evaluate agents for binding to Dectin-1 may be carried out byin vitro using purified or recombinant Dectin-1. Assays can also becarried out in vitro using cells which express Dectin-1—such as liverleukocytes or hepatic stellate cells. Further, screening test may becarried out in vivo using animal models. The cells in culture may beprimary cells or may be secondary cells or cell lines. Examples ofsuitable cells include liver leukocytes (such as dendritic cells,macrophages, CD14⁺ monocytic cells and the like), and hepatic stellatecells. The cells may be enriched from sources such as whole blood. Forexample, whole blood may be obtained from an individual and desiredtypes of leukocytes may be isolated using well known techniques or usingcommercially available kits (such as kits from Miltenyi Biotec). In oneembodiment, the cells may be modified cells. For example, the cells maybe engineered to express or overexpress Dectin-1. The cells in culturecan be maintained by using routine cell culture reagents and procedures.In one embodiment, the assays may be carried out in animals includingmice after administration of Thioacetamide (TAA) or Carbontetrachloride.

The compounds for testing may be part of a library or may be newlysynthesized. Further, the compounds may be purified, partially purifiedor may be present as cell extracts, crude mixtures and the like—i.e.,unpurified. While it is ideal to test each compound separately, acombination of compounds may also be tested.

Dosages of a microglial rescuing agent can vary between wide limits,depending upon the disease or disorder to be treated, the age andcondition of the subject to be treated. In an embodiment where acomposition comprising a microglial rescuing agent is contacted with asample, the concentration of a microglial rescuing agent may be fromabout 0.1 μM to about 40 μM. Alternatively, the concentration of amicroglial rescuing agent may be from about 5 μM to about 25 μM. Forexample, the concentration of a microglial rescuing agent may be about0.1, about 0.25, about 0.5, about 0.6, about 0.7, about 0.8, about 0.9,about 1, about 2.5, about 5, about 6, about 7, about 8, about 9, about10, about 11, about 12, about 13, about 14, about 15, about 16, about17, about 18, about 19, about 20, about 21, about 22, about 23, about24, about 25, about 30, about 35, or about 40 μM. Additionally, theconcentration of a microglial rescuing agent may be greater than 40 μM.For example, the concentration of a microglial rescuing agent may beabout 40, about 45, about 50, about 55, about 60, about 65, about 70,about 75, about 80, about 85, about 90, about 95, or about 100 μM.

In an embodiment where the composition comprising a microglial rescuingagent is administered to a subject, the dose of a microglial rescuingagent may be from about 0.1 mg/kg to about 500 mg/kg. For example, thedose of a microglial rescuing agent may be about 0.1 mg/kg, about 0.5mg/kg, about 1 mg/kg, about 5 mg/kg, about 10 mg/kg, about 15 mg/kg,about 20 mg/kg, or about 25 mg/kg. Alternatively, the dose of amicroglial rescuing agent may be about 25 mg/kg, about 50 mg/kg, about75 mg/kg, about 100 mg/kg, about 125 mg/kg, about 150 mg/kg, about 175mg/kg, about 200 mg/kg, about 225 mg/kg, or about 250 mg/kg.Additionally, the dose of a microglial rescuing agent may be about 300mg/kg, about 325 mg/kg, about 350 mg/kg, about 375 mg/kg, about 400mg/kg, about 425 mg/kg, about 450 mg/kg, about 475 mg/kg or about 500mg/kg.

The amount of a composition described herein that can be combined with apharmaceutically acceptable carrier to produce a single dosage form willvary depending upon the host treated and the particular mode ofadministration. It will be appreciated by those skilled in the art thatthe unit content of agent contained in an individual dose of each dosageform need not in itself constitute a therapeutically effective amount,as the necessary therapeutically effective amount could be reached byadministration of a number of individual doses.

Toxicity and therapeutic efficacy of compositions described herein canbe determined by standard pharmaceutical procedures in cell cultures orexperimental animals for determining the LD50 (the dose lethal to 50% ofthe population) and the ED50, (the dose therapeutically effective in 50%of the population). The dose ratio between toxic and therapeutic effectsis the therapeutic index that can be expressed as the ratio LD50/ED50,where larger therapeutic indices are generally understood in the art tobe optimal.

The specific therapeutically effective dose level for any particularsubject will depend upon a variety of factors including the disorderbeing treated and the severity of the disorder; activity of the specificcompound employed; the specific composition employed; the age, bodyweight, general health, sex and diet of the subject; the time ofadministration; the route of administration; the rate of excretion ofthe composition employed; the duration of the treatment; drugs used incombination or coincidental with the specific compound employed; andlike factors well known in the medical arts (see e.g., Koda-Kimble etal. (2004) Applied Therapeutics: The Clinical Use of Drugs, LippincottWilliams & Wilkins, ISBN 0781748453; Winter (2003) Basic ClinicalPharmacokinetics, 4th ed., Lippincott Williams & Wilkins, ISBN0781741475; Sharqel (2004) Applied Biopharmaceutics & Pharmacokinetics,McGraw-Hill/Appleton & Lange, ISBN 0071375503). For example, it is wellwithin the skill of the art to start doses of the composition at levelslower than those required to achieve the desired therapeutic effect andto gradually increase the dosage until the desired effect is achieved.If desired, the effective daily dose may be divided into multiple dosesfor purposes of administration. Consequently, single dose compositionsmay contain such amounts or submultiples thereof to make up the dailydose. It will be understood, however, that the total daily usage of thecompounds and compositions of the present disclosure will be decided byan attending physician within the scope of sound medical judgment.

(c) Components of the Composition

The present disclosure also provides pharmaceutical compositions. Thepharmaceutical composition comprises a microglial rescuing agent, as anactive ingredient, and at least one pharmaceutically acceptableexcipient.

The pharmaceutically acceptable excipient may be a diluent, a binder, afiller, a buffering agent, a pH modifying agent, a disintegrant, adispersant, a preservative, a lubricant, taste-masking agent, aflavoring agent, or a coloring agent. The amount and types of excipientsutilized to form pharmaceutical compositions may be selected accordingto known principles of pharmaceutical science.

In each of the embodiments described herein, a composition of theinvention may optionally comprise one or more additional drug ortherapeutically active agent in addition to the microglial rescuingagent. In some embodiments, the additional drug or therapeuticallyactive agent is used to treat central nervous system diseases, ordisorders. Other active agents which can be administered together with amicroglial rescuing agent include but are not limited toneurotransmitters, neurotransmitter agonists or antagonists, steroids,corticosteroids (such as prednisone or methyl prednisone)immunomodulating agents (such as beta-interferon), immunosuppressiveagents (such as cyclophosphamide or azathioprine), nucleotide analogs,endogenous opioids, or other currently clinically used drugs. In someembodiments, the secondary agent is selected from a corticosteroid, anon-steroidal anti-inflammatory drug (NSAID), an intravenousimmunoglobulin, a tyrosine kinase inhibitor, a fusion protein, amonoclonal antibody directed against one or more pro-inflammatorycytokines, a chemotherapeutic agent and a combination thereof. In someembodiments, the secondary agent may be a glucocorticoid, acorticosteroid, a non-steroidal anti-inflammatory drug (NSAID), aphenolic antioxidant, an anti-proliferative drug, a tyrosine kinaseinhibitor, an anti IL-5 or an IL5 receptor monoclonal antibody, an antiIL-13 or an anti IL-13 receptor monoclonal antibody, an IL-4 or an IL-4receptor monoclonal antibody, an anti IgE monoclonal antibody, amonoclonal antibody directed against one or more pro-inflammatorycytokines, a TNF-α inhibitor, a fusion protein, a chemotherapeutic agentor a combination thereof. In some embodiments, the secondary agent is ananti-inflammatory drug. In some embodiments, anti-inflammatory drugsinclude, but are not limited to, alclofenac, alclometasone dipropionate,algestone acetonide, alpha amylase, amcinafal, amcinafide, amfenacsodium, amiprilose hydrochloride, anakinra, anirolac, anitrazafen,apazone, balsalazide disodium, bendazac, benoxaprofen, benzydaminehydrochloride, bromelains, broperamole, budesonide, carprofen,cicloprofen, cintazone, cliprofen, clobetasol propionate, clobetasonebutyrate, clopirac, cloticasone propionate, cormethasone acetate,cortodoxone, curcumin, deflazacort, desonide, desoximetasone,dexamethasone dipropionate, diclofenac potassium, diclofenac sodium,diflorasone diacetate, diflumidone sodium, diflunisal, difluprednate,diftalone, dimethyl sulfoxide, drocinonide, endrysone, enlimomab,enolicam sodium, epirizole, etodolac, etofenamate, felbinac, fenamole,fenbufen, fenclofenac, fenclorac, fendosal, fenpipalone, fentiazac,flazalone, fluazacort, flufenamic acid, flumizole, flunisolide acetate,flunixin, flunixin meglumine, fluocortin butyl, fluorometholone acetate,fluquazone, flurbiprofen, fluretofen, fluticasone propionate,furaprofen, furobufen, halcinonide, halobetasol propionate, halopredoneacetate, ibufenac, ibuprofen, ibuprofen aluminum, ibuprofen piconol,ilonidap, indomethacin, indomethacin sodium, indoprofen, indoxole,intrazole, isoflupredone acetate, isoxepac, isoxicam, ketoprofen,lofemizole hydrochloride, lomoxicam, loteprednol etabonate, lysofylline,meclofenamate sodium, meclofenamic acid, meclorisone dibutyrate,mefenamic acid, mesalamine, meseclazone, methylprednisolone suleptanate,mom iflumate, nabumetone, naproxen, naproxen sodium, naproxol, nimazone,olsalazine sodium, orgotein, orpanoxin, oxaprozin, oxyphenbutazone,paranyline hydrochloride, pentosan polysulfate sodium, phenbutazonesodium glycerate, piroxicam, piroxicam cinnamate, piroxicam olamine,pirprofen, prednazate, prifelone, prodolic acid, proquazone, proxazole,proxazole citrate, rimexolone, romazarit, salcolex, salnacedin,salsalate, sanguinarium chloride, seclazone, sermetacin, sudoxicam,sulindac, suprofen, talmetacin, talniflumate, talosalate, tebufelone,tenidap, tenidap sodium, tenoxicam, tesicam, tesimide, tetrydamine,tiopinac, tixocortol pivalate, tolmetin, tolmetin sodium, triclonide,triflumidate, zidometacin, zomepirac sodium, aspirin (acetylsalicylicacid), salicylic acid, corticosteroids, glucocorticoids, tacrolimus,pimecorlimus, mepolizumab, prodrugs thereof, and a combination thereof.

(i) Diluent

In one embodiment, the excipient may be a diluent. The diluent may becompressible (i.e., plastically deformable) or abrasively brittle.Non-limiting examples of suitable compressible diluents includemicrocrystalline cellulose (MCC), cellulose derivatives, cellulosepowder, cellulose esters (i.e., acetate and butyrate mixed esters),ethyl cellulose, methyl cellulose, hydroxypropyl cellulose,hydroxypropyl methylcellulose, sodium carboxymethylcellulose, cornstarch, phosphated corn starch, pregelatinized corn starch, rice starch,potato starch, tapioca starch, starch-lactose, starch-calcium carbonate,sodium starch glycolate, glucose, fructose, lactose, lactosemonohydrate, sucrose, xylose, lactitol, mannitol, malitol, sorbitol,xylitol, maltodextrin, and trehalose. Non-limiting examples of suitableabrasively brittle diluents include dibasic calcium phosphate (anhydrousor dihydrate), calcium phosphate tribasic, calcium carbonate, andmagnesium carbonate.

(ii) Binder

In another embodiment, the excipient may be a binder. Suitable bindersinclude, but are not limited to, starches, pregelatinized starches,gelatin, polyvinylpyrrolidone, cellulose, methylcellulose, sodiumcarboxymethylcellulose, ethylcellulose, polyacrylam ides,polyvinyloxoazolidone, polyvinylalcohols, C₁₂-C₁₈ fatty acid alcohol,polyethylene glycol, polyols, saccharides, oligosaccharides,polypeptides, oligopeptides, and combinations thereof.

(iii) Filler

In another embodiment, the excipient may be a filler. Suitable fillersinclude, but are not limited to, carbohydrates, inorganic compounds, andpolyvinylpyrrolidone. By way of non-limiting example, the filler may becalcium sulfate, both di- and tri-basic, starch, calcium carbonate,magnesium carbonate, microcrystalline cellulose, dibasic calciumphosphate, magnesium carbonate, magnesium oxide, calcium silicate, talc,modified starches, lactose, sucrose, mannitol, or sorbitol.

(iv) Buffering Agent

In still another embodiment, the excipient may be a buffering agent.Representative examples of suitable buffering agents include, but arenot limited to, phosphates, carbonates, citrates, tris buffers, andbuffered saline salts (e.g., Tris buffered saline or phosphate bufferedsaline).

(v) pH Modifier

In various embodiments, the excipient may be a pH modifier. By way ofnon-limiting example, the pH modifying agent may be sodium carbonate,sodium bicarbonate, sodium citrate, citric acid, or phosphoric acid.

(vi) Disintegrant

In a further embodiment, the excipient may be a disintegrant. Thedisintegrant may be non-effervescent or effervescent. Suitable examplesof non-effervescent disintegrants include, but are not limited to,starches such as corn starch, potato starch, pregelatinized and modifiedstarches thereof, sweeteners, clays, such as bentonite,micro-crystalline cellulose, alginates, sodium starch glycolate, gumssuch as agar, guar, locust bean, karaya, pecitin, and tragacanth.Non-limiting examples of suitable effervescent disintegrants includesodium bicarbonate in combination with citric acid and sodiumbicarbonate in combination with tartaric acid.

(vii) Dispersant

In yet another embodiment, the excipient may be a dispersant ordispersing enhancing agent. Suitable dispersants may include, but arenot limited to, starch, alginic acid, polyvinylpyrrolidones, guar gum,kaolin, bentonite, purified wood cellulose, sodium starch glycolate,isoamorphous silicate, and microcrystalline cellulose.

(viii) Excipient

In another alternate embodiment, the excipient may be a preservative.Non-limiting examples of suitable preservatives include antioxidants,such as BHA, BHT, vitamin A, vitamin C, vitamin E, or retinyl palmitate, citric acid, sodium citrate; chelators such as EDTA or EGTA; andantimicrobials, such as parabens, chlorobutanol, or phenol.

(ix) Lubricant

In a further embodiment, the excipient may be a lubricant. Non-limitingexamples of suitable lubricants include minerals such as talc or silica;and fats such as vegetable stearin, magnesium stearate, or stearic acid.

(x) Taste-Masking Agent

In yet another embodiment, the excipient may be a taste-masking agent.Taste-masking materials include cellulose ethers; polyethylene glycols;polyvinyl alcohol; polyvinyl alcohol and polyethylene glycol copolymers;monoglycerides or triglycerides; acrylic polymers; mixtures of acrylicpolymers with cellulose ethers; cellulose acetate phthalate; andcombinations thereof.

(xi) Flavoring Agent

In an alternate embodiment, the excipient may be a flavoring agent.Flavoring agents may be chosen from synthetic flavor oils and flavoringaromatics and/or natural oils, extracts from plants, leaves, flowers,fruits, and combinations thereof.

(xii) Coloring Agent

In still a further embodiment, the excipient may be a coloring agent.Suitable color additives include, but are not limited to, food, drug andcosmetic colors (FD&C), drug and cosmetic colors (D&C), or external drugand cosmetic colors (Ext. D&C).

The weight fraction of the excipient or combination of excipients in thecomposition may be about 99% or less, about 97% or less, about 95% orless, about 90% or less, about 85% or less, about 80% or less, about 75%or less, about 70% or less, about 65% or less, about 60% or less, about55% or less, about 50% or less, about 45% or less, about 40% or less,about 35% or less, about 30% or less, about 25% or less, about 20% orless, about 15% or less, about 10% or less, about 5% or less, about 2%,or about 1% or less of the total weight of the composition.

Controlled-release (or sustained-release) preparations may be formulatedto extend the activity of the agent(s) and reduce dosage frequency.Controlled-release preparations can also be used to effect the time ofonset of action or other characteristics, such as blood levels of theagent, and consequently affect the occurrence of side effects.Controlled-release preparations may be designed to initially release anamount of an agent(s) that produces the desired therapeutic effect, andgradually and continually release other amounts of the agent to maintainthe level of therapeutic effect over an extended period of time. Inorder to maintain a near-constant level of an agent in the body, theagent can be released from the dosage form at a rate that will replacethe amount of agent being metabolized or excreted from the body. Thecontrolled-release of an agent may be stimulated by various inducers,e.g., change in pH, change in temperature, enzymes, water, or otherphysiological conditions or molecules.

(d) Administration

(i) Dosage Forms

The composition can be formulated into various dosage forms andadministered by a number of different means that will deliver atherapeutically effective amount of the active ingredient. Suchcompositions can be administered orally (e.g. inhalation), parenterally,or topically in dosage unit formulations containing conventionalnontoxic pharmaceutically acceptable carriers, adjuvants, and vehiclesas desired. Topical administration may also involve the use oftransdermal administration such as transdermal patches or iontophoresisdevices. The term parenteral as used herein includes subcutaneous,intravenous, intramuscular, intra-articular, or intrasternal injection,or infusion techniques. Formulation of drugs is discussed in, forexample, Gennaro, A. R., Remington's Pharmaceutical Sciences, MackPublishing Co., Easton, Pa. (18th ed, 1995), and Liberman, H. A. andLachman, L., Eds., Pharmaceutical Dosage Forms, Marcel Dekker Inc., NewYork, N.Y. (1980). In a specific embodiment, a composition may be a foodsupplement or a composition may be a cosmetic.

Solid dosage forms for oral administration include capsules, tablets,caplets, pills, powders, pellets, and granules. In such solid dosageforms, the active ingredient is ordinarily combined with one or morepharmaceutically acceptable excipients, examples of which are detailedabove. Oral preparations may also be administered as aqueoussuspensions, elixirs, or syrups. For these, the active ingredient may becombined with various sweetening or flavoring agents, coloring agents,and, if so desired, emulsifying and/or suspending agents, as well asdiluents such as water, ethanol, glycerin, and combinations thereof. Foradministration by inhalation, the compounds are delivered in the form ofan aerosol spray from pressured container or dispenser which contains asuitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer.

For parenteral administration (including subcutaneous, intradermal,intravenous, intramuscular, intra-articular and intraperitoneal), thepreparation may be an aqueous or an oil-based solution. Aqueoussolutions may include a sterile diluent such as water, saline solution,a pharmaceutically acceptable polyol such as glycerol, propylene glycol,or other synthetic solvents; an antibacterial and/or antifungal agentsuch as benzyl alcohol, methyl paraben, chlorobutanol, phenol,thimerosal, and the like; an antioxidant such as ascorbic acid or sodiumbisulfite; a chelating agent such as etheylenediaminetetraacetic acid; abuffer such as acetate, citrate, or phosphate; and/or an agent for theadjustment of tonicity such as sodium chloride, dextrose, or apolyalcohol such as mannitol or sorbitol. The pH of the aqueous solutionmay be adjusted with acids or bases such as hydrochloric acid or sodiumhydroxide. Oil-based solutions or suspensions may further comprisesesame, peanut, olive oil, or mineral oil. The compositions may bepresented in unit-dose or multi-dose containers, for example sealedampoules and vials, and may be stored in a freeze-dried (lyophilized)condition requiring only the addition of the sterile liquid carried, forexample water for injections, immediately prior to use. Extemporaneousinjection solutions and suspensions may be prepared from sterilepowders, granules, and tablets.

For topical (e.g., transdermal or transmucosal) administration,penetrants appropriate to the barrier to be permeated are generallyincluded in the preparation. Pharmaceutical compositions adapted fortopical administration may be formulated as ointments, creams,suspensions, lotions, powders, solutions, pastes, gels, sprays,aerosols, or oils. In some embodiments, the pharmaceutical compositionis applied as a topical ointment or cream. When formulated in anointment, the active ingredient may be employed with either a paraffinicor a water-miscible ointment base. Alternatively, the active ingredientmay be formulated in a cream with an oil-in-water cream base or awater-in-oil base. Pharmaceutical compositions adapted for topicaladministration to the eye include eye drops wherein the activeingredient is dissolved or suspended in a suitable carrier, especiallyan aqueous solvent. Pharmaceutical compositions adapted for topicaladministration in the mouth include lozenges, pastilles, and mouthwashes. Transmucosal administration may be accomplished through the useof nasal sprays, aerosol sprays, tablets, or suppositories, andtransdermal administration may be via ointments, salves, gels, patches,or creams as generally known in the art.

In certain embodiments, a composition comprising a microglial rescuingagent is encapsulated in a suitable vehicle to either aid in thedelivery of the compound to target cells, to increase the stability ofthe composition, or to minimize potential toxicity of the composition.As will be appreciated by a skilled artisan, a variety of vehicles aresuitable for delivering a composition of the present invention.Non-limiting examples of suitable structured fluid delivery systems mayinclude nanoparticles, liposomes, microemulsions, micelles, dendrimers,and other phospholipid-containing systems. Methods of incorporatingcompositions into delivery vehicles are known in the art.

In one alternative embodiment, a liposome delivery vehicle may beutilized. Liposomes, depending upon the embodiment, are suitable fordelivery of a microglial rescuing agent in view of their structural andchemical properties. Generally speaking, liposomes are sphericalvesicles with a phospholipid bilayer membrane. The lipid bilayer of aliposome may fuse with other bilayers (e.g., the cell membrane), thusdelivering the contents of the liposome to cells. In this manner, themicroglial rescuing agent may be selectively delivered to a cell byencapsulation in a liposome that fuses with the targeted cell'smembrane.

Liposomes may be comprised of a variety of different types ofphosolipids having varying hydrocarbon chain lengths. Phospholipidsgenerally comprise two fatty acids linked through glycerol phosphate toone of a variety of polar groups. Suitable phospholids includephosphatidic acid (PA), phosphatidylserine (PS), phosphatidylinositol(PI), phosphatidylglycerol (PG), diphosphatidylglycerol (DPG),phosphatidylcholine (PC), and phosphatidylethanolamine (PE). The fattyacid chains comprising the phospholipids may range from about 6 to about26 carbon atoms in length, and the lipid chains may be saturated orunsaturated. Suitable fatty acid chains include (common name presentedin parentheses) n-dodecanoate (laurate), n-tretradecanoate (myristate),n-hexadecanoate (palm itate), n-octadecanoate (stearate), n-eicosanoate(arachidate), n-docosanoate (behenate), n-tetracosanoate (lignocerate),cis-9-hexadecenoate (palm itoleate), cis-9-octadecanoate (oleate),cis,cis-9,12-octadecandienoate (linoleate), all cis-9, 12,15-octadecatrienoate (linolenate), and allcis-5,8,11,14-eicosatetraenoate (arachidonate). The two fatty acidchains of a phospholipid may be identical or different. Acceptablephospholipids include dioleoyl PS, dioleoyl PC, distearoyl PS,distearoyl PC, dimyristoyl PS, dimyristoyl PC, dipalmitoyl PG, stearoyl,oleoyl PS, palmitoyl, linolenyl PS, and the like.

The phospholipids may come from any natural source, and, as such, maycomprise a mixture of phospholipids. For example, egg yolk is rich inPC, PG, and PE, soy beans contains PC, PE, PI, and PA, and animal brainor spinal cord is enriched in PS. Phospholipids may come from syntheticsources too. Mixtures of phospholipids having a varied ratio ofindividual phospholipids may be used. Mixtures of differentphospholipids may result in liposome compositions having advantageousactivity or stability of activity properties. The above mentionedphospholipids may be mixed, in optimal ratios with cationic lipids, suchas N-(1-(2,3-dioleolyoxy)propyl)-N,N,N-trimethyl ammonium chloride,1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchloarate,3,3′-deheptyloxacarbocyanine iodide,1,1′-dedodecyl-3,3,3′,3′-tetramethylindocarbocyanine perchloarate,1,1′-dioleyl-3,3,3′,3′-tetramethylindo carbocyanine methanesulfonate,N-4-(delinoleylaminostyryl)-N-methylpyridinium iodide, or1,1,-dilinoleyl-3,3,3′,3′-tetramethylindocarbocyanine perchloarate.

Liposomes may optionally comprise sphingolipids, in which spingosine isthe structural counterpart of glycerol and one of the one fatty acids ofa phosphoglyceride, or cholesterol, a major component of animal cellmembranes. Liposomes may optionally contain pegylated lipids, which arelipids covalently linked to polymers of polyethylene glycol (PEG). PEGsmay range in size from about 500 to about 10,000 daltons.

Liposomes may further comprise a suitable solvent. The solvent may be anorganic solvent or an inorganic solvent. Suitable solvents include, butare not limited to, dimethylsulfoxide (DMSO), methylpyrrolidone,N-methylpyrrolidone, acetronitrile, alcohols, dimethylformamide,tetrahydrofuran, or combinations thereof.

Liposomes carrying a microglial rescuing agent may be prepared by anyknown method of preparing liposomes for drug delivery, such as, forexample, detailed in U.S. Pat. Nos. 4,241,046; 4,394,448; 4,529,561;4,755,388; 4,828,837; 4,925,661; 4,954,345; 4,957,735; 5,043,164;5,064,655; 5,077,211; and 5,264,618, the disclosures of which are herebyincorporated by reference in their entirety. For example, liposomes maybe prepared by sonicating lipids in an aqueous solution, solventinjection, lipid hydration, reverse evaporation, or freeze drying byrepeated freezing and thawing. In a preferred embodiment the liposomesare formed by sonication. The liposomes may be multilamellar, which havemany layers like an onion, or unilamellar. The liposomes may be large orsmall. Continued high-shear sonication tends to form smaller unilamellarliposomes.

As would be apparent to one of ordinary skill, all of the parametersthat govern liposome formation may be varied. These parameters include,but are not limited to, temperature, pH, concentration of the microglialrescuing agent, concentration and composition of lipid, concentration ofmultivalent cations, rate of mixing, presence of and concentration ofsolvent.

In another embodiment, a composition of the invention may be deliveredto a cell as a microemulsion. Microemulsions are generally clear,thermodynamically stable solutions comprising an aqueous solution, asurfactant, and “oil.” The “oil” in this case, is the supercriticalfluid phase. The surfactant rests at the oil-water interface. Any of avariety of surfactants are suitable for use in microemulsionformulations including those described herein or otherwise known in theart. The aqueous microdomains suitable for use in the inventiongenerally will have characteristic structural dimensions from about 5 nmto about 100 nm. Aggregates of this size are poor scatterers of visiblelight and hence, these solutions are optically clear. As will beappreciated by a skilled artisan, microemulsions can and will have amultitude of different microscopic structures including sphere, rod, ordisc shaped aggregates. In one embodiment, the structure may bemicelles, which are the simplest microemulsion structures that aregenerally spherical or cylindrical objects. Micelles are like drops ofoil in water, and reverse micelles are like drops of water in oil. In analternative embodiment, the microemulsion structure is the lamellae. Itcomprises consecutive layers of water and oil separated by layers ofsurfactant. The “oil” of microemulsions optimally comprisesphospholipids. Any of the phospholipids detailed above for liposomes aresuitable for embodiments directed to microemulsions. The microglialrescuing agent may be encapsulated in a microemulsion by any methodgenerally known in the art.

In yet another embodiment, a microglial rescuing agent may be deliveredin a dendritic macromolecule, or a dendrimer. Generally speaking, adendrimer is a branched tree-like molecule, in which each branch is aninterlinked chain of molecules that divides into two new branches(molecules) after a certain length. This branching continues until thebranches (molecules) become so densely packed that the canopy forms aglobe. Generally, the properties of dendrimers are determined by thefunctional groups at their surface. For example, hydrophilic end groups,such as carboxyl groups, would typically make a water-soluble dendrimer.Alternatively, phospholipids may be incorporated in the surface of adendrimer to facilitate absorption across the skin. Any of thephospholipids detailed for use in liposome embodiments are suitable foruse in dendrimer embodiments. Any method generally known in the art maybe utilized to make dendrimers and to encapsulate compositions of theinvention therein. For example, dendrimers may be produced by aniterative sequence of reaction steps, in which each additional iterationleads to a higher order dendrimer. Consequently, they have a regular,highly branched 3D structure, with nearly uniform size and shape.Furthermore, the final size of a dendrimer is typically controlled bythe number of iterative steps used during synthesis. A variety ofdendrimer sizes are suitable for use in the invention. Generally, thesize of dendrimers may range from about 1 nm to about 100 nm.

(II) Methods

The present disclosure encompasses a method of modulating microglialactivity in a subject or in a sample, the method generally comprisingcontacting the subject or sample with a composition comprising aneffective amount of a microglial rescuing agent. In another aspect, thepresent disclosure encompasses a method of modulating microglialmetabolism in a subject in need thereof, the method generally comprisingadministering to the subject a composition comprising a therapeuticallyeffective amount of a microglial rescuing agent. In yet another aspect,the present disclosure provides a composition comprising of a microglialrescuing agent for use in vitro, in vivo, or ex vivo. In someembodiments, the present invention provides a method of treatingmicroglial dysfunction in a subject having a microglialdysfunction-associated neurodegenerative disease comprisingadministering to a subject a therapeutically effective amount of amicroglial rescuing agent. One aspect of the present disclosure providesfor a treatment of a subject with a neurodegenerative disease (e.g., AD)with creatine, a creatine analog, or Dectin-1 agonist as a treatment toenhance microglial responses by sustaining cell metabolism inindividuals with single nucleotide polymorphisms (SNPs) or othermutations affecting microglial functions. Suitable compositionscomprising a microglial rescuing agent are disclosed herein, forinstance those described in Section I.

Provided is a process of treating a neurological disease, disorder, orcondition associated with microglial dysfunction in a subject in needadministration of a therapeutically effective amount of a microglialrescuing agent, so as to enhance microglial function, inhibit aneurological disease, disorder, or condition associated with microglialdysfunction, slow the progress of a neurological disease, disorder, orcondition associated with microglial dysfunction, or limit thedevelopment of a neurological disease, disorder, or condition associatedwith microglial dysfunction.

There has been an every growing expansion of understanding of theinvolvement of microglia in central nervous system (CNS) disorders. Ahost of new molecular tools and mouse models of disease are increasinglyimplicating this enigmatic type of nervous system cell as a key playerin conditions ranging from neurodevelopmental disorders such as autismto neurodegenerative disorders such as Alzheimer's disease and chronicpain. Contemporaneously, diverse roles are emerging for microglia in thehealthy brain, from sculpting developing neuronal circuits to guidinglearning-associated plasticity. The term “glial cell”, as used hereinrefers to connective tissue cells of the central nervous systemproviding structural and functional support to the neuronal cells of thecentral nervous system, including, for example, in the form of providingnutrition and homeostasis and/or by participation in signal transmissionin the nervous system. Glial cells include, but are not limited to,astrocytes (also referred to herein as astroglial cells), microglia, andoligodendrocytes.

The term “microglial cell” or “microglia”, as used herein, refers to aclass of glial cells involved in the mediation of an immune responsewithin the central nervous system by acting as macrophages. Microglialcells are capable of producing exosomes, and further include differentforms of microglial cells, including amoeboid microglial cells, ramifiedmicroglial cells and reactive microglial cells. Microglial cells includereactive microglia, which are defined as quiescent ramified microgliathat transform into a reactive, macrophage-like state and accumulate atsites of brain injury and inflammation to assist in tissue repair andneural regeneration.

One aspect of the present disclosure provides for a treatment of asubject with a microglial-dysfunction associated disease or disorder.The microglial-dysfunction associated disease or disorder may be anycentral nervous system disease or disorder in which disrupted microglialfunction contributes to pathology or symptoms. In non-limiting examples,microglial-dysfunction associated diseases and disorders includeAlzheimer's disease, Parkinson's disease, Nasu-Hakola disease, priondiseases, multiple sclerosis, HIV-dementia, amyotrophic lateralsclerosis (ALS), frontal temporal dementia, neuropathic pain, and autismspectrum disorders. For example, microglial-dysfunction associateddiseases and disorders include those described in Salter and Stevens,Nature Medicine volume 23, pages 1018-1027 (2017), the description ofwhich is incorporated herein by reference. In some embodiments, themicroglial-dysfunction associated disease or disorder is AD. In oneaspect, the microglial-dysfunction associated disease or disorder isassociated with mutations in TREM2 or ApoE.

As shown herein, a subject with a neurodegenerative disease (e.g., AD)or a microglial-dysfunction associated disease in a subject with ApoE orTREM2 variants can be treated with a microglial rescuing agent(optionally in combination with conventional treatments). It has beenshown that creatine and analogs can provide improved neuroprotection insubjects with ApoE or TREM2 variant compared to a subject with the samedisease without the ApoE or TREM2 variant.

Surprisingly, the present disclosure provides for the identification ofa specific subset of patients with mutations in ApoE or TREM2 as havingan improved response to a microglial rescuing agent. For example, FIG. 6presents data showing that the creatine treatment in TREM2 knock out ADmouse model works surprisingly or unexpectedly better than in the ADmouse model without the TREM2 knock out.

Elevated risk of developing Alzheimer's disease (AD) is associated withhypomorphic variants of TREM2, a surface receptor required formicroglial responses to neurodegeneration, including proliferation,survival, clustering and phagocytosis. How TREM2 promotes such diverseresponses is unknown. Here, we find that microglia in AD patientscarrying TREM2 risk variants and TREM2-deficient mice with AD-likepathology have abundant autophagic vesicles, as do TREM2-deficientmacrophages under growth factor limitation or ER stress. Combinedmetabolomics and RNA-seq linked this anomalous autophagy to defectivemTOR signaling, which affects ATP levels and biosynthetic pathways.Metabolic derailment and autophagy were offset in vitro throughDectin-1, a receptor that elicits TREM2-like intracellular signals, andcyclocreatine, a creatine analog that can supply ATP. Dietarycyclocreatine markedly tempered autophagy, restored microglialclustering around plaques, and decreased plaque-adjacent neuronaldystrophy in TREM2-deficient mice with amyloid-β pathology. Thus, TREM2enables microglial responses during AD by sustaining cellular energeticand biosynthetic metabolism.

Several creatine analogs have been used in vitro and in vivo tosupplement deficiency in TREM2, which can play an anti-inflammatory rolein the pathogenesis of Alzheimer's. Microglia of mice and humansdeficient in Trem-2 undergo increased autophagy in response to stressessuch as plaques associated with AD. Treatment of Trem-2 deficientmacrophages with nicotinamide mononucleotide, cyclocreatine, andphosphocreatine was able to rescue metabolic activity and preventautophagy and cell death. In addition, excessive neuronal damage wasreversed in vivo when the compounds were administered to Trem-2deficient 5XFAD mice. The invention can be utilized to decrease therisk/severity of AD in patients carrying mutations in ApoE or Trem2 thatlimit the function of microglial cells.

According to an aspect of the invention a pharmaceutical compositioncomprising a microglial rescuing agent is used for modulating microglialactivity. The method generally comprises contacting a microglia with apharmaceutical composition comprising a microglial rescuing agent. Insome embodiments, the method comprising contacting a microglia in vivoby administering a pharmaceutical composition comprising a microglialrescuing agent. Microglial activity can be measured by cell viability,mTOR signaling, presence/absence of autophagy, Syk signaling, PI3-Ksignaling, microgliosis, microglial clustering, neurite dystrophy, andmicroglial metabolism. Standard techniques and assays may be used tomeasure microglial activity including those described in the examplesbelow. In some embodiments, microglial activity is measured by cellviability, wherein increased cell viability indicates increasedmicroglial activity. In some embodiments, microglial activity ismeasured by mTOR signaling, wherein increased mTOR signaling indicatesincreased microglial activity. In some embodiments, microglial activityis measured by the presence of autophagy, wherein decreased autophagyindicates increased microglial activity. In some embodiments, contactinga microglia with a pharmaceutical composition comprising a microglialrescuing agent results in increased microglial activity relative to anuntreated control.

In certain aspects, a therapeutically effective amount of a compositionof the invention may be administered to a subject. Methods describedherein are generally performed on a subject in need thereof. A subjectin need of the therapeutic methods described herein can be a subjecthaving, diagnosed with, suspected of having, or at risk for developing aneurological disease, disorder, or condition associated with microglialdysfunction. A determination of the need for treatment will typically beassessed by a history and physical exam consistent with the disease orcondition at issue. Diagnosis of the various conditions treatable by themethods described herein is within the skill of the art. Administrationis performed using standard effective techniques, including peripherally(i.e. not by administration into the central nervous system) or locallyto the central nervous system. Peripheral administration includes but isnot limited to oral, inhalation, intravenous, intraperitoneal,intra-articular, subcutaneous, pulmonary, transdermal, intramuscular,intranasal, buccal, sublingual, or suppository administration. Localadministration, including directly into the central nervous system (CNS)includes but is not limited to via a lumbar, intraventricular orintraparenchymal catheter or using a surgically implanted controlledrelease formulation. The route of administration may be dictated by thedisease or condition to be treated. For example, if the disease orcondition is COPD or IPF, the composition may be administered viainhalation. Alternatively, is the disease or condition isosteoarthritis, the composition may be administered via intra-articularinvention. It is within the skill of one in the art, to determine theroute of administration based on the disease or condition to be treated.In a specific embodiment, a composition of the invention is administeredorally.

Pharmaceutical compositions for effective administration aredeliberately designed to be appropriate for the selected mode ofadministration, and pharmaceutically acceptable excipients such ascompatible dispersing agents, buffers, surfactants, preservatives,solubilizing agents, isotonicity agents, stabilizing agents, and thelike are used as appropriate. Remington's Pharmaceutical Sciences, MackPublishing Co., Easton Pa., 16Ed ISBN: 0-912734-04-3, latest edition,incorporated herein by reference in its entirety, provides a compendiumof formulation techniques as are generally known to practitioners.

For therapeutic applications, a therapeutically effective amount of acomposition of the invention is administered to a subject. A“therapeutically effective amount” is an amount of the therapeuticcomposition sufficient to produce a measurable response. In variousembodiments, an effective amount of a microglial rescuing agentdescribed herein can substantially enhance microglial function, inhibita neurological disease, disorder, or condition associated withmicroglial dysfunction, slow the progress of a neurological disease,disorder, or condition associated with microglial dysfunction, or limitthe development of a neurological disease, disorder, or conditionassociated with microglial dysfunction. Actual dosage levels of activeingredients in a therapeutic composition of the invention can be variedso as to administer an amount of the active compound(s) that iseffective to achieve the desired therapeutic response for a particularsubject. The selected dosage level will depend upon a variety of factorsincluding the activity of the therapeutic composition, formulation, theroute of administration, combination with other drugs or treatments,age, the age-related disease or condition, the degenerative disease, thefunction-decreasing disorder, the symptoms, and the physical conditionand prior medical history of the subject being treated. In someembodiments, a minimal dose is administered, and dose is escalated inthe absence of dose-limiting toxicity. Determination and adjustment of atherapeutically effective dose, as well as evaluation of when and how tomake such adjustments, are known to those of ordinary skill in the artof medicine.

The frequency of dosing may be daily or once, twice, three times, ormore per day, per week or per month, as needed as to effectively treatthe symptoms. The timing of administration of the treatment relative tothe disease itself and duration of treatment will be determined by thecircumstances surrounding the case. Treatment could begin immediately,such as at the site of the injury as administered by emergency medicalpersonnel. Treatment could begin in a hospital or clinic itself, or at alater time after discharge from the hospital or after being seen in anoutpatient clinic. Duration of treatment could range from a single doseadministered on a one-time basis to a life-long course of therapeutictreatments.

Typical dosage levels can be determined and optimized using standardclinical techniques and will be dependent on the mode of administration.

A subject may be a rodent, a human, a livestock animal, a companionanimal, or a zoological animal. In one embodiment, the subject may be arodent, e.g. a mouse, a rat, a guinea pig, etc. In another embodiment,the subject may be a livestock animal. Non-limiting examples of suitablelivestock animals may include pigs, cows, horses, goats, sheep, llamasand alpacas. In still another embodiment, the subject may be a companionanimal. Non-limiting examples of companion animals may include pets suchas dogs, cats, rabbits, and birds. In yet another embodiment, thesubject may be a zoological animal. As used herein, a “zoologicalanimal” refers to an animal that may be found in a zoo. Such animals mayinclude non-human primates, large cats, wolves, and bears. In apreferred embodiment, the subject is a human.

Definitions

When introducing elements of the present disclosure or the preferredaspects(s) thereof, the articles “a,” “an,” “the,” and “said” areintended to mean that there are one or more of the elements. The terms“comprising,” “including,” and “having” are intended to be inclusive andmean that there may be additional elements other than the listedelements.

As used herein, the following definitions shall apply unless otherwiseindicated. For purposes of this invention, the chemical elements areidentified in accordance with the Periodic Table of the Elements, CASversion, and the Handbook of Chemistry and Physics, 75^(th) Ed. 1994.Additionally, general principles of organic chemistry are described in“Organic Chemistry,” Thomas Sorrell, University Science Books,Sausalito: 1999, and “March's Advanced Organic Chemistry,” 5^(th) Ed.,Smith, M. B. and March, J., eds. John Wiley & Sons, New York: 2001, theentire contents of which are hereby incorporated by reference.

In some embodiments, numbers expressing quantities of ingredients,properties such as molecular weight, reaction conditions, and so forth,used to describe and claim certain embodiments of the present disclosureare to be understood as being modified in some instances by the term“about.” In some embodiments, the term “about” is used to indicate thata value includes the standard deviation of the mean for the device ormethod being employed to determine the value. In some embodiments, thenumerical parameters set forth in the written description and attachedclaims are approximations that can vary depending upon the desiredproperties sought to be obtained by a particular embodiment. In someembodiments, the numerical parameters should be construed in light ofthe number of reported significant digits and by applying ordinaryrounding techniques. Notwithstanding that the numerical ranges andparameters setting forth the broad scope of some embodiments of thepresent disclosure are approximations, the numerical values set forth inthe specific examples are reported as precisely as practicable. Thenumerical values presented in some embodiments of the present disclosuremay contain certain errors necessarily resulting from the standarddeviation found in their respective testing measurements. The recitationof ranges of values herein is merely intended to serve as a shorthandmethod of referring individually to each separate value falling withinthe range. Unless otherwise indicated herein, each individual value isincorporated into the specification as if it were individually recitedherein.

In some embodiments, the terms “a” and “an” and “the” and similarreferences used in the context of describing a particular embodiment(especially in the context of certain of the following claims) can beconstrued to cover both the singular and the plural, unless specificallynoted otherwise. In some embodiments, the term “or” as used herein,including the claims, is used to mean “and/or” unless explicitlyindicated to refer to alternatives only or the alternatives are mutuallyexclusive.

The terms “comprise,” “have” and “include” are open-ended linking verbs.Any forms or tenses of one or more of these verbs, such as “comprises,”“comprising,” “has,” “having,” “includes” and “including,” are alsoopen-ended. For example, any method that “comprises,” “has” or“includes” one or more steps is not limited to possessing only those oneor more steps and can also cover other unlisted steps. Similarly, anycomposition or device that “comprises,” “has” or “includes” one or morefeatures is not limited to possessing only those one or more featuresand can cover other unlisted features.

All methods described herein can be performed in any suitable orderunless otherwise indicated herein or otherwise clearly contradicted bycontext. The use of any and all examples, or exemplary language (e.g.“such as”) provided with respect to certain embodiments herein isintended merely to better illuminate the present disclosure and does notpose a limitation on the scope of the present disclosure otherwiseclaimed. No language in the specification should be construed asindicating any non-claimed element essential to the practice of thepresent disclosure.

EXAMPLES

The following examples are included to demonstrate various embodimentsof the present disclosure. It should be appreciated by those of skill inthe art that the techniques disclosed in the examples that followrepresent techniques discovered by the inventors to function well in thepractice of the invention, and thus can be considered to constitutepreferred modes for its practice. However, those of skill in the artshould, in light of the present disclosure, appreciate that many changescan be made in the specific embodiments which are disclosed and stillobtain a like or similar result without departing from the spirit andscope of the invention.

Introduction

As shown herein, the present disclosure provides for the use of amicroglial rescuing agent that can increase microglial energeticmetabolism in patients with mutations in genes which increase the oddsfor the development of Alzheimer's disease by effecting microglialfunction i.e. ApoE, Trem2. Microglial rescuing agents such as NMN,cyclocreatine, and phosphocreatine have been used in vitro and in thecase of cyclocreatine in vivo to supplement Trem2-deficient cells ormice with AD-like pathology (5XFAD mice).

As shown herein, microglia in Trem2-deficient mice and humans carryingdisease associated SNPs in TREM2 are less energetically competent andexhibit increased autophagy in response to stress such as the pathologicstate associated with Alzheimer's disease. Supplementation of media withwild-type and Trem2-deficient bone marrow derived macrophages with NMN,cyclocreatine, and phosphocreatine were able to rescue Trem2-deficientcells metabolic capacity and prevent autophagy and cell death. In invivo studies in 5XFAD and Trem2-deficient 5XFAD mice we show that we canincrease microgliosis, microglial clustering around plaques, decreasemicroglial autophagy, decrease microglial cell death, and subsequentlydecrease neuronal dystrophy around plaques.

As shown herein, LC3 positive puncta was observed in microglia inindividuals carrying disease associated SNPs in TREM2 and that thesepuncta are reminiscent of the LC3 puncta in microglia in 5XFAD mice.Trem2-deficient and ApoE-deficient mice have similar microglial responseto plaque deposition. Cyclocreatine treatment of Trem2-deficient 5XFADprevented the formation of autophagic vesicles and cell death whilereversing excessive neuronal damage. This invention could treat andpotentially normalize the risk for the development of Alzheimer'sdisease and limit disease severity in individuals carrying mutationswhich compromise microglial responsiveness to amyloid β plaques inAlzheimer's. Such genes include ApoE and Trem2.

Electron and confocal microscopy was used to analyze microglia from5XFAD mice, which develop AR accumulation that mimics AD pathology dueto the expression of mutant APP and PS1 under neural-specific elementsof the mouse Thy1 promoter. Microglia from 5XFAD mice lacking TREM2 hadmany more autophagic vesicles than did microglia in SXFAD mice. Thisobservation was replicated in humans, as microglia in AD patientscarrying TREM2 risk variants also had more autophagic vesicles than didmicroglia in AD patients with the common TREM2 variant. Autophagy is anintracellular degradation pathway essential for cellular and energyhomeostasis (Galluzzi et al., 2014). It provides a mechanism for theelimination of misfolded proteins and damaged organelles and compensatesfor nutrient deprivation during cell starvation through recycling ofcytosolic components. Because autophagy is partially regulated bymammalian target of rapamycin (mTOR)-dependent pathways (Saxton andSabatini, 2017), the impact of TREM2-deficiency on mTOR activation wasassessed and found that, indeed, anomalous autophagy reflected defectiveactivation of mTOR signaling. Similarly, enhanced autophagy was observedin TREM2-deficient macrophages in vitro, which was further amplified bygrowth-factor limitation or endoplasmic reticulum (ER) stress; thisprovided a model system for probing biochemical and metabolic pathwaysin microglia during Aβ accumulation. Combined metabolomics, RNAsequencing (RNA-seq), and system analyses of TREM2-deficient macrophagesconfirmed the impairment of mTOR activation, energetic pathways, ATPlevels, and biosynthetic pathways. Thus, TREM2 sustains cell energeticand biosynthetic metabolism through mTOR signaling. Metabolic derailmentand autophagy were offset in vitro through activation of Dectin-1, asurface receptor that triggers a signaling pathway similar to that ofTREM2-DAP12 (Dambuza and Brown, 2015). Metabolic abnormalities were alsorescued by incubating cells with the creatine analog1-carboxymethyl-2-iminoimidazolidine (cyclocreatine), which canpassively cross membranes and, upon phosphorylation by creatine kinase,generate a supply of ATP for energy demands independent of theTREM2-mTOR axis (Kurosawa et al. 2012). Remarkably, dietaryadministration of cyclocreatine in 5XFAD mice lacking TREM2 preventedmicroglial autophagy, enhanced microglia numbers and clustering aroundAβ plaques, and mitigated plaque-associated neurite dystrophy. Thisprovides proof of principle that strategies aimed at sustaining basicmicroglial metabolism may be promising for treatment of AD and otherneurodegenerative diseases associated with microglial dysfunction.

Example 1: Defect in TREM2 Elicits Autophagy In Vivo in the 5XFAD MouseModel and AD Patients

To determine the impact of TREM2 deficiency on microglia function, wedirectly examined the structure of microglia from the 5XFAD mouse modelof AD by transmission electron microscopy (TEM). Strikingly, microgliafrom Trem2^(−/− 5)XFAD mice contained abundantmultivesicular/multilamellar structures suggestive of autophagosomes,which were largely absent in microglia from 5XFAD, Trem2^(−/−) orwild-type (WT) mice (FIG. 1A, FIG. 1B). To determine whether thesestructures reflected ongoing autophagy in situ, we examined brainsections by confocal microscopy for the presence of LC3⁺ puncta, whichdenote autophagosomes decorated by lipidated LC3II (Klionsky et al.,2016). Many large LC3⁺ puncta were evident in microglia inTrem2^(−/− 5)XFAD mice, whereas LC3⁺ puncta were sparse in microglia inWT, Trem2^(−/−), and 5XFAD mice (FIG. 1C, FIG. 1D and FIG. 8).Remarkably, these observations were translatable to human disease. Weobserved dramatically more LC3⁺ microglia in post-mortem brain sectionsfrom both R47H and R62H heterozygous AD patients than in those fromcase-matched AD patients homozygous for the common TREM2 variant (FIG.1E, FIG. 1F, Table 1). Taken together, these data suggest thatautophagic-like vesicles accumulate in the microglia of TREM2-deficientmice and humans with TREM2 risk alleles during the development of AD.

TABLE 1 Characteristics of Human Tissue Donors (Related to FIG. 1) CDR(Est. at TREM2 Sex Age (yrs) Braak Stage TOD) CERAD Status TREM2 VariantCarrier Female 93.98 N.A. 3 N.D. R47H/CV Male 74.85 V 2 Definite R47H/CVMale 83.75 N.A. 3 N.D. R47H/CV Male 90.64 V 3 Definite R47H/CV Female88.22 IV 3 Definite R47H/CV Male 85.64 V 3 Definite R47H/CV Male 78.23 V3 Definite R47H/CV Female 83.16 N.A. 3 N.D. R62H/CV Female 89.31 VI 3Definite R62H/CV Female 89.37 N.A. 3 N.D. R62H/CV Female 78.98 N.A. 3N.D. R62H/CV Case- Matched Control Female 95.73 VI 3 Definite CV/CV Male71.6 VI 3 Definite CV/CV Male 87.57 V 3 Definite CV/CV Male 91.08 V 1Definite CV/CV Female 84.47 V 3 Definite CV/CV Male 80.97 VI 3 DefiniteCV/CV Female 85.43 N.A. 3 N.D. CV/CV Female 81.26 VI 3 Definite CV/CV

Example 2: TREM 2 Deficiency Impairs mTOR Signaling and Enhances AMPKActivation in Microglia

To corroborate the association between TREM2 deficiency and increasedautophagy, we performed biochemical analyses on sorted microglia exvivo. The ratio of lipidated LC3II to non-lipidated LC3I was markedlyhigher in microglia from Trem2^(−/−) 5XFAD mice than in microglia from5XFAD mice, consistent with the increased number of autophagic vesiclesin TREM2-deficient microglia. To determine whether the increase inautophagosomes was due to activation of autophagy or blockade oflysosomal degradation, we measured protein and mRNA levels of p62, anautophagy cargo protein that is digested by lysosomal enzymes. Theamount of p62 protein was lower in microglia from Trem2^(−/−) 5XFAD micethan in microglia from 5XFAD mice. This difference was unrelated totranscription, as p62 (Sqstm1) mRNA levels were similar. Thus, TREM2deficiency results in bona fide autophagy (FIG. 2A, FIG. 2B and FIG.9A).

Why is autophagic flux amplified in 5XFAD mice lacking TREM2? Autophagyoften reflects an adaptive response to stress that can occur when cellscannot satisfy increased demands for energy and protein synthesis(Kroemer et al., 2010). Since the serine/threonine kinase target ofrapamycin (mTOR) has a crucial role in stimulating both energetic andanabolic metabolism, cell growth and proliferation (Laplante andSabatini, 2012), we hypothesized that the autophagy observed inmicroglia in Trem2^(−/−) 5XFAD mice might result from a defect in mTORsignaling. mTOR signals through two distinct complexes, mTORC1 andmTORC2. Immunoblotting of sorted microglia from Trem2^(−/−) 5XFAD and5XFAD mice revealed decreased phosphorylation of 4EBP1, an mTORC1effector, as well as AKT at serine 473 and NDRG1, both mTORC2 effectors,in the TREM2-deficient microglia (FIG. 2A). Ulk1, a key inducer ofautophagy, which is inhibited by mTOR signaling through phosphorylationat serine 757 (Kim et al., 2011), was less phosphorylated in microgliafrom Trem2^(−/−) 5XFAD mice, consistent with reduced mTOR activation andincreased autophagy. Impaired mTOR signaling was associated withphosphorylation of AMP-activated protein kinase (AMPK), a sensor of lowenergy states (FIG. 2A).

Providing further evidence for defective energetic and anabolicmetabolism associated with TREM2 deficiency, microglia from Trem2^(−/−)5XFAD mice had a lower mitochondrial mass than did microglia from 5XFADmice (FIG. 2C, FIG. 2D). Furthermore, gene expression microarrayanalyses of sorted microglia from Trem2^(−/−) 5XFAD and 5XFAD micerevealed decreased expression of genes encoding translation initiationfactors, ribosomal proteins, glucose transporters, glycolytic enzymes,as well as the transcription factor HIF1α that controls glycolysis inTREM2-deficient microglia (FIG. 9B-FIG. 9G). Taken together, these datademonstrate that during the development of AD, TREM2 deficiency derailsthe mTOR pathway, anabolic and energetic metabolism in microglia, whichinduces compensatory autophagy in both mice and humans.

To determine whether autophagy in TREM2-deficient microglia caneffectively compensate for the metabolic defects in vivo and preventapoptosis, we examined brain sections by confocal microscopy for thepresence of cleaved caspase-3, an indicator of apoptosis. We found thatcleaved caspase-3 was much more abundant in microglia in Trem2^(−/−)5XFAD mice than in microglia in 5XFAD mice (FIG. 2E, FIG. 2F).Additionally, the percentage of microglia with colocalization of cleavedcaspase-3 and LC3⁺ puncta was higher in Trem2^(−/−) 5XFAD mice than in5XFAD mice (FIG. 9H, FIG. 9I). Thus, autophagy may not be sufficient tosustain the microglial response to stress, at least at the late timepoint of disease progression analyzed.

Example 3: Macrophages Lacking TREM2 Inadequately Signal Through mTORand Undergo More Autophagy

We then asked whether TREM2-deficiency could derail mTOR signaling inbone marrow-derived macrophages (BMDMs) from WT and Trem2^(−/−) mice invitro. To mimic the metabolic stress that occurs during disease, we usedgrowth factor deprivation. BMDMs were cultured overnight inconcentrations of CSF1-containing L cell-conditioned medium (LCCM)ranging from optimal to limiting (10% to 0.5%). Trem2^(−/−) BMDMscontained more autophagic vesicles (FIG. 3A, FIG. 3B) and had a higherLC3II/LC3I ratio than did WT cells when CSF-1 was limiting (FIG. 3C).Addition of the lysosomal inhibitor bafilomycin greatly increased LC3IIin Trem2^(−/−) BMDMs, confirming that the increase in autophagosomes wasdue to increased autophagic flux rather than reduced autophagosomedegradation (FIG. 3D, FIG. 3E).

As observed in sorted microglia, autophagy in BMDMs was linked toimpaired mTOR signaling. Trem2^(−/−) BMDMs had less phosphorylated4EBP1, 473S AKT and NDRG1 (FIG. 3F) and more phosphorylated AMPK (FIG.3G) in both optimal CSF1 and limiting CSF1 than did WT BMDMs. Inlimiting CSF1, Trem2^(−/−) BMDMs had decreased inhibitoryphosphorylation of Ulk1 at serine 757, while activating phosphorylationof Ulk1 at serine 317 increased (FIG. 3H). Thus, lack of TREM2suppressed mTOR activation and elicited compensatory AMPK and Ulk1activation and autophagy in BMDMs in response to metabolic stress, verysimilar to our observations of microglia in 5XFAD mice.

As TREM2 signaling adapters DAP12 and DAP10 have been shown to activatePI3-K, which in turn can activate mTOR, we asked whether enhanced mTORsignaling in WT BMDM compared to Trem2^(−/−) BMDM was dependent onPI3-K. Inhibition of PI3-K with wortmannin or LY294002 caused a majorreduction in phosphorylation of mTOR and its downstream targets in WTBMDMs; the residual amount of phosphorylation was similar to that seenin Trem2^(−/−) BMDMs (FIG. 3I and FIG. 10A). In addition to limitingCSF1, other stressors may differentially modulate mTOR signaling andautophagy in WT and Trem2^(−/−) BMDMs. Treatment with tunicamycin, whichprovokes endoplasmic reticulum (ER) stress, the unfolded proteinresponse and autophagy, induced greater LC3II/LC3I ratios and less AktS473 phosphorylation in Trem2^(−/−) BMDMs than in WT BMDMs (FIG. 3J,FIG. 3K). Thus, TREM2 deficiency affects cell responses to multiplestressors.

TREM2 signaling may not just have a more pronounced effect on cellsunder stress conditions, but may actually increase under suchconditions. When cultured in media containing 10% FBS, TREM2 reportercells, which express GFP upon TREM2 engagement, showed some activation(FIG. 10B) (Wang et al., 2015). However, upon serum starvation, asignificantly higher proportion of reporter cells became activated,possibly due to exposure of the TREM2 ligand phosphatidylserine on theouter leaflet of stressed cells. This activation could be blocked byinclusion of an anti-TREM2 antibody. Thus, in multiple in vitro settingsof stress, TREM2-expressing cells are more able to sustain mTORactivation and suppress autophagy in a PI3K-dependent manner than arecells lacking TREM2.

Example 4: TREM2 Deficiency Curtails Anabolic and Energetic Metabolismin BMDMs

To directly demonstrate the impact of TREM2 deficiency on energetic andanabolic pathways in BMDMs, we performed mass spectrometry to quantifycellular metabolites and RNA sequencing (RNA-seq) to quantify mRNAlevels of metabolic enzymes. Analysis of metabolite data alone, or incombination with RNA-Seq data by a systems-based algorithm, revealedwidespread differences between WT and Trem2^(−/−) BMDMs in variousmetabolic pathways (Sergushichev et al., 2016). Compared to WT BMDMs inoptimal CSF-1, Trem2^(−/−) BMDMs cultured under the same conditionsexhibited: 1) a marked decrease of key intermediates in the synthesis ofnucleotides (e.g. phosphoribosyl pyrophosphate), N-glycosylated proteins(e.g. UDP-glucose), and phospholipids (e.g. CDP-ethanolamine); 2) adecrease in glycolytic metabolites (e.g. glucose 6-phosphate andfructose bisphosphate) and tricarboxylic acid (TCA) cycle intermediates(citrate and succinate); and 3) an increase in catabolic products ofamino acids (e.g. indolacetate) and phospholipid precursors (e.g.glycerol 3-phosphate) (FIG. 4A, FIG. 11A). Moreover, a selectiveincrease in malate and fumarate suggested an enhanced malate-aspartateshuttle to sustain defective NADH oxidation and NAD regeneration (FIG.4A). Unbiased network analysis combining metabolic and RNA-seq datahighlighted defects in metabolites and enzymes involved in glycolysis,TCA cycle and pentose phosphate pathway in Trem2^(−/−) BMDMs (FIG. 4B).

CSF-1 reduction further deteriorated energy and anabolic metabolism inTrem2^(−/−) BMDMs. Under these conditions, again in comparison to WTBMDMs, Trem2^(−/−) BMDMs underwent a marked increase in symmetricaldimethyl arginine, indicative of protein catabolism, as well as anincrease in ADP-ribose, indicative of NAD degradation (FIG. 4C, FIG.11B-FIG. 11C). Furthermore, stores of high-energy phosphates, such asphosphocreatine and ATP, were depleted in Trem2^(−/−) BMDMs cultured inlimiting CSF1 conditions (FIG. 11D). A luciferase-based ATP assayconfirmed an ATP deficiency in Trem2^(−/−) BMDMs, which was exacerbatedat low CSF1 concentrations (FIG. 4D). We further assessed the energymetabolism of WT and Trem2^(−/−) BMDMs using the Seahorse analyzer. Alower extracellular acidification rate (ECAR), indicative of lessglycolytic flux, was noted in Trem2^(−/−) BMDMs both at baseline andafter induction of maximal glycolytic capacity by oligomycin and FCCP.This deficit widened relative to WT cells as the CSF1 concentration wasreduced (FIG. 4E). Trem2 BMDMs had only a slightly reduced oxygenconsumption rate (OCR) compared to WT BMDMs when cultured in standardCSF1 concentrations, indicating relatively intact oxidativephosphorylation; however, a deficit in OCR emerged as the CSF1concentration was reduced (FIG. 4E). Trem2^(−/−) BMDMs also had fewermitochondria than WT BMDMs on a per cell basis as measured byMitoTracker Green fluorescence and by the mitochondrial-to-nuclear DNAratio (FIG. 4F, FIG. 4G). These findings were not restricted to BMDMs,as resting and thioglycolate-elicited peritoneal TREM2-deficientmacrophages also had a lower mitochondrial mass than did WT macrophages(FIG. 11E-FIG. 11H). Cultured adult primary Trem2^(−/−) microgliarecapitulated the deficiencies in energetic metabolism and mTORsignaling as well as autophagy observed in Trem2^(−/−) BMDMs (FIG.11I-FIG. 11M). Thus, lack of TREM2-mTOR signaling impairs the energystatus and anabolism of BMDMs and other primary macrophages both insteady state and under energetic stress.

Example 5: Enhanced Energy Storage or Dectin-1 Signaling can Compensatefor TREM2 Deficiency In Vitro

Given the dramatic effect of TREM2 deficiency on mTOR activation andenergy utilization in BMDMs, we tested whether bypassing TREM2 anddirectly compensating for these deficits by alternative means couldrestore the cellular energy status of Trem2^(−/−) BMDMs. Musclephysiology studies have extensively demonstrated that creatine phosphatecontributes to the regeneration of ATP and to the maintenance ofuniformly high ATP/ADP ratios in muscle fibers (Walker, 1979). Moreover,the creatine analog 1-carboxymethyl-2-iminoimidazolidine (cyclocreatine)can, upon phosphorylation, generate a long-acting phosphagen that caneffectively sustain cellular ATP levels during increased energy demand(Kurosawa et al., 2012; Woznicki and Walker, 1979). Thus, we testedwhether addition of cyclocreatine to the culture medium could rescueenergetic metabolism in Trem2-deficient BMDMs. Indeed, incubation withcyclocreatine improved ECAR, which was accompanied by less autophagy,increased mTOR signaling and viability (FIG. 5A-FIG. 5C and data notshown).

To test whether engagement of receptors that elicit signaling pathwayssimilar to those of TREM2 could also mitigate autophagy and support cellsurvival, Trem2^(−/−) and WT BMDMs were cultured with depleted zymosan,a selective ligand of dectin-1, which activates Syk and PI3K signalingindependent of DAP12 (Dambuza and Brown, 2015). Dectin-1 activationcurbed autophagy in CSF1-starved TREM2-deficient BMDMs to levels seen inWT BMDMs in low CSF-1, as indicated by a reduction in the LC3II/LC3Iratio (FIG. 5D, FIG. 5E) along with increased amounts of p62 (FIG. 5F).Treatment with zymosan also enhanced cellular ATP levels in Trem2^(−/−)BMDMs, restoring them to WT BMDM levels. (FIG. 5G). Thus, alternativeenergetic and signaling pathways can compensate for lack of TREM2signaling.

Example 6: Cyclocreatine Rescues Microgliosis and Clustering andModerates Neurite Dystrophy In Vivo

Because cyclocreatine rescued metabolism and viability and suppressedautophagy in Trem2^(−/−) BMDMs in vitro and given previous studiesshowing that cyclocreatine is passively transported across membranes andcan accumulate and function as a phosphagen in the mouse brain in vivo(Kurosawa et al., 2012), we asked whether dietary supplementation withcyclocreatine could rescue microglial function and suppress autophagy invivo in Trem2^(−/−) 5XFAD mice. The drinking water of 5XFAD andTrem2^(−/−) 5XFAD mice was supplemented with cyclocreatine from 10 weeksof age until 8 months of age. Remarkably, significantly fewermultivesicular/multilamellar structures were seen by TEM in microglia inTrem2^(−/−) 5XFAD mice treated with cyclocreatine than in microglia inuntreated mice (FIG. 6A, FIG. 6B). Corroborating this with confocalmicroscopy, the percentage of LC3⁺ microglia, the number of LC3puncta/cell, and the percentage of cleaved caspase-3⁺ microglia were allsignificantly decreased in Trem2^(−/−) 5XFAD mice treated withcyclocreatine (FIG. 12B and FIG. 6C, FIG. 6E, FIG. 6F,). Furthermore,the number of microglia/high powered field (HPF) in plaque-bearingregions of the cortex and clustering of microglia around plaques wereboth significantly increased (FIG. 6C-FIG. 6D and FIG. 12A) inTrem2^(−/−) 5XFAD mice treated with cyclocreatine compared to untreatedTrem2^(−/−) 5XFAD mice. These findings indicate that dietarysupplementation with cyclocreatine is sufficient to partially rescue thedefect in microgliosis and microglial clustering around plaques inTrem2^(−/−) 5XFAD mice, while concomitantly mitigating autophagy anddeath of the microglia.

To assess the impact of cyclocreatine on microglial activation, which isalso impaired in Trem2^(−/−) 5XFAD mice, we quantified the percentage ofmicroglia that expressed the microglial activation marker osteopontin(Spp1) in 5XFAD and Trem2^(−/−) 5XFAD mice, a protein that, in thebrain, is specifically upregulated in microglia in the context of Aβdeposition (Orre et al., 2014; Wang et al., 2015). Untreated Trem2^(−/−)5XFAD mice had very few Spp1⁺ microglia, while 5XFAD,cyclocreatine-treated 5XFAD, and cyclocreatine-treated Trem2^(−/−) 5XFADmice all had significantly more Spp1⁺ microglia (FIG. 7A, FIG. 7B).Moreover, biochemical analysis of microglia isolated ex vivodemonstrated that cyclocreatine treatment of Trem2^(−/− 5)XFAD mice alsorestored microglial mTOR signaling and significantly limited autophagycompared to untreated Trem2^(−/−) 5XFAD mice (FIG. 7C, FIG. 7D).

As a major function of TREM2 in vivo is enabling microglia to form abarrier around plaques that prevents spreading of Aβ fibrils andalleviates dystrophy of plaque-adjacent neurites (Wang et al., 2016;Yuan et al., 2016), we asked whether cyclocreatine treatment ofTrem2^(−/−) 5XFAD mice impacted plaque morphology and/or neuronaldystrophy. While plaques in untreated Trem2^(−/−) 5XFAD mice had a lowerdensity than those in 5XFAD mice as measured by methoxy-X04 stainingintensity, the density of plaques in cyclocreatine treated Trem2^(−/−)5XFAD mice resembled that of plaques in 5XFAD mice (FIG. 7E), althoughplaque shape complexity was not significantly altered (FIG. 12K).Despite reducing plaque density, cyclocreatine did not moderate plaqueaccumulation or the engulfment of plaque particulates by microglia, atleast at this time point (FIG. 12F-FIG. 12K). As APP is known toaccumulate in dystrophic neurites, we used APP deposition in distinctrounded particles around plaques to assess neurite dystrophy (Masliah etal., 1996; Wang et al., 2016; Yuan et al., 2016). Cyclocreatinetreatment of Trem2^(−/−) 5XFAD mice significantly reducedplaque-associated neurite dystrophy compared to untreated Trem2^(−/−)5XFAD mice to levels observed in 5XFAD mice (FIG. 7F, FIG. 7G). Takentogether, these data indicate that cyclocreatine administration improvesmicroglial metabolism and the protective response to Aβ plaques inTREM2-deficient 5XFAD mice.

Discussion

Increasing evidence supports the hypothesis that the microglial responseto AD lesions controls disease progression (Gold and El Khoury, 2015;Hong et al., 2016; Meyer-Luehmann and Prinz, 2015; Perry and Holmes,2014; Tejera and Heneka, 2016; Wang et al., 2016; Yuan et al., 2016).Toll-like receptors and NOD-like receptors have been previouslyimplicated in the microglia response to Aβ accumulation and shown tomediate an inflammatory response that contributes to pathology (Freemanand Ting, 2016; Heneka et al., 2015; Heneka et al., 2013). To sustaincytokine secretion, these receptors induce a striking metabolicreprogramming, which consists of a switch from fatty acid metabolism andoxidative phosphorylation to glycolysis (O'Neill and Pearce, 2016). Inour study, TREM2 emerges as an innate immune receptor that impactsmicroglia metabolism in AD through a distinct mechanism, which consistsof basic activation of mTOR signaling that supports long-term celltrophism, survival, growth, and proliferation, rather than drasticmetabolic reprogramming. This function of TREM2 is reminiscent of thetonic function of the B cell antigen receptor in mature B cells, whichdelivers survival signals through PI3-K (Werner et al., 2010). Likewise,cell membrane phospholipids and lipoprotein particles may continuouslyengage TREM2, inducing tonic mTOR signaling through upstream activators,such as PI3-K, PDK1 and AKT, which are recruited by the TREM2-associatedsignaling subunits DAP12 and DAP10 (Ford and McVicar, 2009; Peng et al.,2010). This concept provides a unifying mechanism to explain thereported broad and long-term impact of TREM2 on diverse microglialfunctions, such as survival, proliferation, clustering around plaques,as well as phagocytosis of apoptotic cells and myelin debris (Jay etal., 2017; Neumann and Takahashi, 2007; Wang et al., 2015; Yeh et al.,2016; Yuan et al., 2016).

We found that the defective mTOR signaling in TREM2-deficient microgliais associated with a compensatory increase of autophagy in vitro and invivo in AD. Reduced glycolysis and autophagy are known to attenuateinflammation (Netea-Maier et al., 2016) and, indeed, microglia from5XFAD mice lacking TREM2 weakly express inflammatory mediators incomparison to microglia from 5XFAD mice (Wang et al., 2015). Moreover,autophagy may also enhance microglial clearance of Aβ (Cho et al., 2014;Lucin et al., 2013; Shibuya et al., 2014), as it does in neurons (Naraet al., 2006; Komatsu et al., 2006; Yang et al., 2011). However, along-term defect in mTOR activation results in global microglialdysfunction, reduced cell viability and proliferation, as demonstratedby increased caspase-3 activation in microglia and by the previouslyreported increase in dying microglia around plaques in Trem2^(−/−) 5XFADmice (Wang et al., 2015). Thus, while increased autophagy may bebeneficial in reducing inflammation and Aβ load in the short-term, adefect in mTOR signaling is detrimental and severely impairs microgliafitness and capacity to respond to Aβ accumulation in the long-term.

TREM2-deficient microglia have long been thought to improperly remain ina homeostatic state during neurodegenerative disease rather thanresponding appropriately to pathology, a paradigm that has beensupported by transcriptomic analysis of these cells. However, for thefirst time, we demonstrate that on a biochemical and ultrastructurallevel, TREM2-deficient microglia adopt a severely divergent cellularstate that does not reflect homeostasis during neurodegeneration, with adramatic loss of mTOR signaling and robust induction of autophagy. Theseresults suggest that TREM2-deficient microglia are not simply ignoringplaque pathology, but rather that they are being actively driven into astressed state that is normally compensated by TREM2-dependent survivalsignals. An important implication of this finding is that microglia in aneurodegenerative environment probably receive not only positiveactivating signals but also negative cytotoxic signals. Thus, it may notbe microglial activation per se that is required to protect againstneurodegeneration, but rather avoidance of a dysfunctional, low-energystate induced by the disease. Based on our findings, previous reports ofimpaired microglial activation in a variety of settings may be due toeither impaired recognition of activating signals or to impairedresistance to cytotoxic signals—two possibilities that can bedistinguished by the strength of mTOR signaling. Counteracting suchdysfunction by metabolic compensation may also represent a fundamentallydistinct therapeutic approach.

Along these lines, our study shows that the defect in mTOR-mediatedmetabolic activation in TREM2-deficient cells can be corrected in vitrothrough the creatine kinase pathway or by triggering the dectin-1pathway, which transmits intracellular signals similar to those ofTREM2. Based on these results, we adopted a therapeutic strategy basedon the use of cyclocreatine, an analog of creatine that crossesmembranes, enters the brain (Woznicki and Walker, 1979), can bephosphorylated and dephosphorylated by creatine kinases (McLaughlin etal., 1972), and can generate a supply of ATP (Kurosawa et al., 2012).Remarkably, we found that administration of dietary cyclocreatinethroughout the progression of Aβ accumulation improves microgliaviability, numbers and clustering around Aβ plaques. As a result,plaques are denser and, most importantly, plaque-associated neuritedystrophy is greatly reduced. Although cyclocreatine treatment was notsufficient to reduce the overall Aβ plaque accumulation, this may dependon time point chosen for analysis and/or cyclocreatine dosage andduration of treatment. While the creatine kinase pathway has beenpreviously recognized to play an important role in the CNS inneurotransmitter release, membrane potential maintenance, Ca²⁺homeostasis, and ion gradient restoration (Snow and Murphy, 2001; Wyssand Kaddurah-Daouk, 2000), our results indicate that this system mayalso be exploited for sustaining microglial metabolism. It should benoted that in certain settings cyclocreatine can inhibit creatine kinaseand can also have systemic effects such as alteration in pancreatichormones and glucose metabolism (Ara et al., 1998; Kuiper et al., 2008).As this is the case and creatine and creatine analogs like cyclocreatineare available over the counter for use we must emphasize that thesefindings are the result of proof of principal studies and we do notadvise the use cyclocreatine as a preventative treatment for AD. Futurestudies will be required to precisely define the mechanisms throughwhich cyclocreatine impacts microglial responses to Aβ. Additionally, itwill be important to determine whether cyclocreatine has any impact onproteolytic shedding of TREM2 from microglia, which results in therelease of soluble TREM2 with potential pro-survival functions.Altogether, our study provides proof of principle that strategies aimedat sustaining microglial metabolism may be promising for therapeuticintervention in AD and other neurodegenerative diseases linked to TREM2deficiency and microglial dysfunction in general.

Experimental Model and Subject Details

Mice.

Mice were of mixed sexes. Mice within experiments were age and sexmatched. For studies using 5XFAD and Trem2^(−/−) 5XFAD animals allanimals were 8 months of age at the time of use. For bone marrow andprimary microglia mice were used from 6 weeks of age until 12 weeks ofage. Mice used in this study include WT C57BL/6J, 5XFAD, Trem2^(−/−),and Trem2^(−/−) 5XFAD animals. All animals were backcrossed until atleast >98% C57BL/6J confirmed by genotype wide microsatellite typing.Mice were housed under specific pathogen free conditions. Mice fromdifferent genotypes were cohoused. Mice did not undergo any proceduresprior to their stated use. For cyclocreatine treatment mixed litters ofsex matched mice were randomly assigned to experimental groups. Allstudies performed on mice were done in accordance with the InstitutionalAnimal Care and Use Committee at Washington University in St. Louisapproved all protocols used in this study.

Human Post-Mortem Samples.

Characteristics of donors of human post-mortem brain tissue at the timeof collection is indicated in Table 1. Samples from 7 R47H, 4 R62H, and8 case matched AD patients were examined. Samples were obtained from theKnight Alzheimer's Disease Research Center at Washington University.Protocol numbers: Healthy Aging and Senile Dementia (NASD) MorphologyCore: 89-0555 and Program Project: Alzheimer's Disease Research Center(ADRC): 89-0556.

Cell Lines and Primary Cells.

Bone marrow derived macrophages and microglia were prepared from sex andage matched mice. To prepare bone marrow-derived macrophages, femurs andtibias were removed and flushed with PBS. Cells were counted and platedat 2.5×10⁶ cells/100 mm petri dish in RPMI supplemented with Glutamax,penicillin/streptomycin, non-essential amino acids, pyruvate, and 10%heat inactivated fetal bovine serum (complete RPMI) and 10% L-cellconditioned medium (LCCM). Cells were cultured for 4-5 days before use.Microglia were prepared as previously described (Wang et al., 2015).Briefly, brains were dissociated by using a Neural Tissue DissociationKit (T) (Miltenyi Biotech Cat. Number 130-093-231). Cells suspensionswere labeled with anti-mouse CD45 magnetic beads and isolated on LScolumns (Miltenyi Biotic). Cells were plated onto poly-L-lysine coatedpolystyrene plates in complete RPMI supplemented with 20% LCCM and 10ng/ml human TGF-β. Media was changed on day 3 post plating and cellswere used 5-7 days post plating.

Trem2 reporter cells were maintained in 10% FBS in RPMI-1640supplemented with sodium pyruvate, GlutaMAX, andpenicillin/streptomycin. Trem2 reporter cells were based on the 2B4NFAT-GFP cells developed by Arase et al. (Arase et al., 2002). The sexof the mouse from which 2B4 t-cell hybridoma cells were derived has notbeen reported (Arase et al., 2002; Hedrick et al., 1982).

Method Details

Mice.

The generation of Trem2^(−/−) and Trem2^(−/−) 5XFAD mice has beendescribed previously (Oakley et al., 2006; Turnbull et al., 2006; Wanget al., 2015). All mice were on a C57BL/6 background. Age and sexmatched mice were used for all experiments; experimental cohorts of micewere cohoused from birth to control for the microbiota. For in vivocyclocreatine treatment 10-week old mice were put oncyclocreatine-containing water, treatment was continued until micereached 8 months of age (Santa Cruz SC-217964 S). Desired intake ofcyclocreatine was approximately 0.28 mg/g of body weight/day, which isapproximately the same as the standard creatine dose used in humans of285 mg/kg of body weight/day (Kurosawa et al., 2012). Cyclocreatine wasadministered in drinking water at a final concentration of 2.33 mg/ml.The Institutional Animal Care and Use Committee at Washington Universityin St. Louis approved all protocols used in this study.

Human Post-Mortem Brain Tissue.

Paraffin-embedded sections (8 um) from the frontal cortex of individualscarrying the common variant (CV) of TREM2 (8) or heterozygous for the CVand either R47H (7), R62H (4) were obtained from the Knight Alzheimer'sDisease Research Center at Washington University. Protocol numbers:Healthy Aging and Senile Dementia (NASD) Morphology Core: 89-0555 andProgram Project: Alzheimer's Disease Research Center (ADRC): 89-0556.R47H and R62H carriers were case matched for age, gender, andCERAD-Reagan plaque score to CV TREM2 control individuals. Detaileddemographic characteristics are provided in Table 1.

Immunohistochemistry of Human Post-Mortem Brain Tissue.

Brain sections were deparaffinized with xylene and rehydrated withdecreasing concentrations of ethanol. Antigen retrieval was performed byincubating sections for 20 minutes in a 95° C. citrate buffer bath (10mM sodium citrate, 0.05% Tween-20, pH 6.0) prior to staining. Sectionswere blocked in 3% goat serum in PBS for 30 minutes at room temperature(RT) followed by incubation with rabbit anti-Iba1 (1:250, Wako; catalogno. 019-19741) overnight at 4° C. Sections were washed in PBS andincubated for 1 hour at room temperature (RT) with methoxy-X04 (20μg/ml) (Tocris Bioscience #4920) and anti-rabbit DyLight 549 (VectorLaboratories DI-1549). Sections were washed and incubated overnight inanti-LC3A/B Alexa Fluor 488 (Cell Signaling Technologies #13082).Sections were washed and mounted using Fluoromount G (SouthernBiotech#0100-01) and images were collected using a Nikon A1Rsi+ confocalmicroscope. Images were then processed with Imaris 7.7 (Bitplane).

Cell Culture and Biochemical Assays.

To prepare bone marrow-derived macrophages, femurs and tibias wereremoved and flushed with PBS. Cells were counted and plated at 2.5×10⁶cells/100 mm petri dish in RPMI supplemented with Glutamax,penicillin/streptomycin, non-essential amino acids, pyruvate, and 10%heat inactivated fetal bovine serum (complete RPMI) and 10% L-cellconditioned medium (LCCM). Cells were cultured for 4-5 days before use.Microglia were prepared as previously described (Wang et al., 2015).Briefly, brains were dissociated by using a Neural Tissue DissociationKit (T) (Miltenyi Biotech Cat. Number 130-093-231). Cell suspensionswere labeled with anti-mouse CD45 magnetic beads and isolated on LScolumns (Miltenyi Biotic). Cells were plated onto poly-L-lysine coatedpolystyrene plates in complete RPMI supplemented with 20% LCCM and 10ng/ml human TGF-β. Media was changed on day 3 post plating and cellswere used 5-7 days post plating. ATP concentrations were determined withan ATP Determination Kit (Invitrogen).

Microglia Sorting.

Microglia were isolated from the indicated animals as previouslydescribed (Wang et al., 2015). CD45⁺, CD11 b⁺, F4/80⁺ (Biolegend Cat.Number 103134, eBioscience Cat. Numbers 11-0112 and 17-4801) cells inthe brain were fluorescence-activated cell-sorted (FACS) directly intoRLT-plus lysis buffer for microarray or 2% FBS in PBS for TEM orimmunoblot lysates. For microarray RNA extraction was performed using aRNeasy micro kit (QIAGEN). Microarray hybridization (Affymetrix MoGene1.0 ST array) and data processing were performed at the WashingtonUniversity Genome Center. For normalization, raw data was processed byRobust Multi-Array (RMA) method and genes were pre-filtered forexpression value 120 expression units, a cut-off above which genes havea 95% chance of expression demonstrated in Immgen data set, which usesthe same array platform (Wang et al., 2015). QIAGEN IPA analysis wasperformed by comparing fold change and p-values for all genes 5XFAD andTrem2-deficient 5XFAD microglia. Heatmaps and hierarchical clusteringwere generated from preselected gene-lists using Morpheus. Microarraydata has been deposited at GEO:GSE65067.

Immunoblotting.

BMDM or microglia were lysed in RIPA buffer (50 mM Tris, 150 mM NaCl, 1%SDS, and 1% Triton X100) containing PMSF, leupeptin, activated sodiumorthovanidate, apoprotinin, and phosphatase inhibitor cocktail 3 (SigmaAldrich Cat. Number P0044). Lysates were flash frozen on dry ice andstored at −80° C. until use. Lysates were thawed and 4×LDS runningbuffer and 10% β-mercaptoethanol were added. Lysates were heated to 95°C. for 10 minutes and run on either a 15% polyacrylamide gel with a 4%stacking gel, a 12% bis-tris gel (Nupage), or a 4-12% bis-tris gel(Nupage). Proteins were transferred to nitrocellulose and blocked for 1hour at RT in 5% milk in Tris buffered saline with 0.05% Tween 20(TBST). Membranes were incubated in primary antibody overnight at 4° C.Membranes were subsequently washed and incubated in Lienco anti-rabbitHRP for 1 hour at RT, washed, and developed using either SuperSignalWest Pico Chemiluminescent Substrate or a combination of SuperSignalWest Pico Chemiluminescent Substrate and SuperSignal West Femto Chemiluminescent Substrate.

Metabolite Profiling by EIS-MS/MS.

BMDMs were cultured in either 0.5% or 10% LCCM overnight in completeRPMI. Polar metabolites were extracted according to General Metabolicsprotocol for extraction of polar metabolites from adherent mammaliancell culture. Briefly, cells were washed in pre-warmed 75 mM ammoniumcarbonate in water. Metabolites were extracted by addition of 70° C. 70%ethanol for 3 minutes. Ethanol was removed and plates were washed withadditional 70° C. 70% ethanol. Debris was pelleted by spinning at 14,000rpm in a tabletop microcentrifuge for 10 minutes at 4° C. Extracts weremoved to a fresh tube and shipped to General Metabolics for assessmentby EIS-MS/MS. Differential expression analysis was done using limma.

RNA-Seq Analysis.

Cells were cultured as described in the metabolite profiling byEIS-MS/MS section above. mRNA was extracted from cell lysates usingoligo-dT beads (Invitrogen). For cDNA synthesis, we used custom oligo-dTprimer with a barcode and adaptor-linker sequence(CCTACACGACGCTCTTCCGATCT-XXXXXXXX-T15)(SEQ ID NO:1). After first-strandsynthesis, samples were pooled together based on Actb qPCR values andRNA-DNA hybrids were degraded with consecutive acid-alkali treatment.Subsequently, a second sequencing linker (AGATCGGAAGAGCACACGTCTG)(SEQ IDNO:2) was ligated with T4 ligase (NEB) followed by SPRI-beads (AgencourtAMPure XP, BeckmanCoulter) clean-up. The mixture was enriched by PCR for12 cycles and SPRI-beads (Agencourt AMPure XP, BeckmanCoulter) purifiedto yield final strand-specific RNA-seq libraries. Libraries weresequenced using a HiSeq 2500 (IIlumina) using 50 bp×25 bp pair-endsequencing. Second read (read-mate) was used for sample demultiplexing.Reads were aligned to the GRCm38.p2 assembly of mouse genome using STARaligner. Aligned reads were quantified using quant3p script to accountfor specifics of 3′ sequencing. RefSeq genome annotation was used andDESeq2 was used for differential gene expression analysis. RNAseq datahas been deposited at GEO:GSE98563.

Network Analysis.

Network analysis was performed as previously described utilizing ShinyGAM (Sergushichev et al., 2016; Vincent et al., 2015). We considered anetwork of chemical mappings between carbon atoms in substrates andproducts for all annotated reactions in KEGG database using RPAIRsentries. The scores for nodes and edges were assigned according tolog(p-value), such that highly significant gene or metabolite signalshad positive scores and not significant had negative scores. Using anexact solver we found a module with a maximal weight, with countingpositive scores maximum once for a measured entity (a mass-spectrometrypeak or a gene). For clarity, addition edges between nodes in the modulewere added if the corresponding gene was highly expressed (was in a top3000 expressed genes).

qRT-PCR.

Total RNA was isolated with TRIzol Reagent (Invitrogen) andsingle-strand cDNA was synthesized with a High Capacity cDNA ReverseTranscription Kit (Applied Biosystems). Genomic DNA was extracted usingthe QIAamp DNA micro kit (Qiagen) to determine mtDNA/nDNA ratios.Real-time PCR was performed using SYBR Green real-time PCR master mix(Thermo-Fisher) and LightCycler 96 detection system (Roche). mtDNAprimers were to cytochrome c oxidase subunit 1 and nDNA primers were toNADH:ubiquinone oxidoreductase core subunit V1.

Metabolism Assays.

For real-time analysis of extracellular acidification rates (ECAR)macrophages were analyzed using an XF96 Extracellular Flux Analyzer(Agilent Technologies). Cells were incubated overnight in complete RPMIin the indicated concentration of LCCM with or without cyclocreatine (10mM). Measurements were taken under basal conditions and following thesequential addition of 1 μM oligomycin and 1.5 μM fluoro-carbonylcyanide phenylhydrazone (FCCP) (purchased from Sigma-Aldrich).

Transmission Electron Microscopy.

For ultrastructural analyses, cells were fixed in 2%paraformaldehyde/2.5% glutaraldehyde in 100 mM sodium cacodylate buffer,pH 7.2 for 1 hr at RT (Polysciences Inc., Warrington, Pa.). Samples werewashed in sodium cacodylate buffer and postfixed in 1% osmium tetroxidefor 1 hr (Polysciences Inc.). Samples were then rinsed extensively indeionized water prior to en bloc staining with 1% aqueous uranyl acetatefor 1 hr (Ted Pella Inc., Redding, Calif.). Following several rinses indH₂O, samples were dehydrated in a graded series of ethanol and embeddedin Eponate 12 resin (Ted Pella Inc.). Sections of 95 nm were cut with aLeica Ultracut UCT ultramicrotome (Leica Microsystems Inc., Bannockburn,Ill.), stained with uranyl acetate and lead citrate, and viewed on aJEOL 1200 EX transmission electron microscope (JEOL USA Inc., Peabody,Mass.) equipped with an AMT 8 megapixel digital camera and AMT ImageCapture Engine V602 software (Advanced Microscopy Techniques, Woburn,Mass.).

For quantitation of multivesicular/multilamellar structures, 30 cellsthat were cross-sectioned through the nucleus (indicating cross-sectionthrough the middle of cell) were randomly chosen, and images of eachcell were taken at 6,000× and 20,000× magnification. The cross-sectionalarea of each of the multivesicular structures were determined usingImage J 1.38 g (National Institutes of Health, USA, customized for AMTimages). Data is expressed as the 1) total number of amultivesicular/multilammelar structures per cross-sectional area ofcytosol and 2) the total cross-sectional area ofmultivesicular/multilamellar structures per area of cytosol.

Preparation of Brain Samples and Confocal Microscopy.

Confocal microscopy analysis was performed as previously described (Wanget al., 2015). Briefly, mice were anesthetized with ketamine/xylazineand perfused with ice-cold PBS containing 1 U/ml of heparin. Brains werefixed in 4% PFA overnight at 4° C. rinsed in PBS and incubated overnightat 4° C. in 30% sucrose before freezing in a 2:1 mixture of 30% sucroseand optimal cutting temperature compound. Serial 40 μm coronal sectionswere cut on a cryo-sliding microtome. Floating sections from 1.1 mmBregma to 0.8 mm Bregma for cortical imaging or slides with fixed humansections were stained with Iba-1 (Waco Chemicals Cat. Number 019-19741)overnight at 4° C. followed by staining with anti-rabbit IgG DyLight 549(Vector Laboratories Cat. Number DI-1549) and methoxy-X04 (Tocris Cat.Number 4920) for 1 hour at RT. Finally, sections were stained for withanti-LC3 Alexa 488±anti-cleaved caspase 3 (Cell Signaling TechnologiesCat. Number 13082 and 9602). Images were collected using a Nikon A1Rsi+confocal microscope. Images were then processed with Imaris 7.7(Bitplane).

Microglia Clustering Analysis.

Positions of microglia and positions and volumes of plaques withinz-stacks were derived from analysis in Imaris, and microglia-plaqueassociation was determined using automated scripts in Matlab. Briefly,each plaque in the z-stack was modeled as an idealized sphere with thesame volume and center of mass. Microglia density within 15 μm of theplaque surface was determined by isolating the voxels of the image thatfall within 15 μm of the edge of the idealized plaque. The number ofmicroglia contained in these voxels was divided by the total volume ofthose voxels to obtain density for a single plaque. Densities of allplaques in a z-stack were averaged together, and the resulting valueswere averaged together for all z-stacks corresponding to a singleanimal.

Reporter Cell Assay.

The 2B4 T cell hybridoma cell line was retrovirally transduced with anNFAT-GFP reporter construct, and TREM2 reporter cells were generated bya second retroviral transduction with a TREM2 overexpression constructand selected by puromycin resistance, as previously described (Wang etal., 2015). Cells were cultured routinely in complete media (10% FBS inRPMI-1640 supplemented with sodium pyruvate, GlutaMAX, andpenicillin/streptomycin). For serum starvation, cells were plated at adensity of 25,000 cells/well in a 96-well plate in either complete mediaor RPMI-1640 in the presence of 20% anti-TREM2 hybridoma supernatant(clone M178, generated in house) or 20% isotype control hybridomasupernatant (Wang et al., 2015). After 16 hours, the percent of GFP+cells among live cells was measured by flow cytometry.

Quantification of Methoxy-X04 Coverage.

To measure total plaque area, brain sections were stained with methoxyX04. Images were collected using a Nikon Eclipse 80i microscope. Forquantitative analysis, images were converted to 8-bit greyscale andstitched using the “Stitching” plugin in ImageJ. Cortex (˜1.1 mm Bregmato 0.8 mm Bregma) and hippocampus (˜−1.7 Bregma to −2.4 Bregma) weredetermined by manual selection. The threshold of selected images wereset at 1.5× mean intensity of the selected area to highlight plaques andanalyzed using the “Measure” function in ImageJ to calculate the percentarea covered. Identified objects after thresholding were individuallyinspected to confirm the object as a plaque or not. Two brain sectionsper mouse were used for quantification. The average of two sections wasused to represent a plaque load for each mouse.

Plaque Morphology Analysis.

Methoxy-X04-stained sections were imaged by confocal microscopy using a60× objective and 1.5× digital zoom in the cortex at ˜1.1 mm Bregma to0.8 mm Bregma. Z images were taken at 1.2 μm intervals. 20-30 μmz-stacks were z-projected by maximum intensity projection and individualplaques were selected in ImageJ. Each individual plaque was traced usinga combination of thresholds and edge detection and smoothened usingimage erosion. The average intensity was determined by averaging valuesof pixels within the plaque trace. The shape index was calculated as4π*(perimeter pixels)²/(all pixels).

Quantification and Statistical Analysis

Data in FIG.s are presented as mean±SEM. Unless otherwise statedstatistical analysis was performed using Prism (GraphPad).Quantification of confocal microscopy, immunoblots, and electronmicroscopy images were performed using Imaris, ImageJ, Matlab, and FIJI.Differential metabolite expression was analyzed using limma. Pathwayanalysis of microarray data was performed using IPA software. RNAseqanalysis was performed by using Second read (read-mate) for sampledemultiplexing. Reads were aligned using STAR aligner and quantifiedusing quant3p script. RefSeq genome annotation was used and DESeq2 wasused for differential gene expression analysis. Combined RNAseq andmetabolite network analysis was performed utilizing Shiny GAM.Statistical analysis to compare the mean values for multiple groups wasperformed using Prism by one-way ANOVA with Holm-Sidak's multiplecomparisons test. Comparison of two groups was performed in Prism usinga two-tailed unpaired t-test (Mann Whitney). Values were accepted assignificant if P≤0.05. Intragroup variation compared between groups wassimilar in all experiments.

Data and Software Availability

Microarray data has been deposited at GEO:GSE65067.

RNAseq data has been deposited at GEO:GSE98563.

TABLE 2 KEY RESOURCES TABLE REAGENT or RESOURCE SOURCE IDENTIFIERAntibodies anti-Pan Actin Cell Signaling Cat# 4968 Technologies anti-p62Cell Signaling Cat# 8025 Technologies anti-phospho Akt 473Cell Signaling Cat# 9271 Technologies anti-phospho Ulk1 317Cell Signaling Cat# 12753 Technologies anti-phospho Ulk1 757Cell Signaling Cat# 6888 Technologies anti-phospho NDRG1 Cell SignalingCat# 3217 Technologies anti-phospho S6K Cell Signaling Cat# 2710Technologies anti-phospho 4EBP1 Cell Signaling Cat# 2855 Technologiesanti-Akt Cell Signaling Cat# 9272 Technologies anti-S6K Cell SignalingCat# 9202 Technologies anti-phospho AMPKα Cell Signaling Cat# 2535Technologies anti-AMPKα Cell Signaling Cat# 5832 Technologies anti-LC3Cell Signaling Cat# 4108 Technologies anti-phospho mTOR 2448Cell Signaling Cat# 2971 Technologies anti-Spp1 R and D Systems Cat#AF808 anti-Xbp-1s BD Biosciences Cat# 562642 anti-Iba1 Wako Cat#019-19741 anti-cleaved caspase-3 Alexa 647 Cell Signaling Cat# 9602Technologies anti-LC3 488 Cell Signaling Cat# 13082 Technologiesanti-APP Milipore Cat# MAB348 anti-CD45 BV421 Biolegend Cat# 103134anti-CD11b FITC eBioscience Cat# 11-0112 anti-F4/80 APC eBioscience Cat#17-4801 anti-Trem2 Colonna Lab M1178 doi: 10.1016/j.ce11.2015. 01.049.anti-rabbit DyLight 549 Vector Laboratories Cat#D1-1549Bacterial and Virus Strains Murine Trem2 expressed in pMXs- Cell Biolabshttps://www.cellbiolabs. IRES-Puro Retroviral Expression com/pmxs-Vector ires-puro-retroviral- expression-vector Biological SamplesHuman brain tissue for Alzheimer's  Knight Alzheimer's DiseaseProtocol numbers: disease patients(TREM2^(CV/CV) (8), Research Center at Healthy Aging andTREM2^(CV/R47H) (7), TREM2^(CV/R62H) (4)) Washington UniversitySenile Dementia (HASD) Morphology Core: 89-0555 and Program Project:Alzheimer's Disease Research Center (ADRC): 89-0556.Chemicals, Peptides, and Recombinant Proteins CyclocreatineSanta Cruz Biotechnology Cat# SC-217964 S Methoxy X04 Tocris BiosciencesCat# 4920 MitoTraker Green Invitrogen Cat# M7514 Wortmannin MilliporeCat# 12-338 Ly294002 Calbiochem Cat# 440202 Tunicamycin Sigma-AldrichCat# 654380 Bafilomycin A1 from Streptomyces Sigma-Aldrich Cat#B1793-10UG griseus ToPro3 Iodide Life Technologies Cat# T3605Zymosan, depleted Invivogen Cat# tlrl-zyd Oligomycin Cayman ChemicalCat# 11341 FCCP Cayman Chemical Cat# 370865 Critical Commercial AssaysATP Assay Invitrogen Cat# A22066 Neural Tissue Dissociation Kit (T)Miltenyi Biotech Cat# 130-093-231 Deposited Data GEO/G5E65067Microarray data GEO/G5E98563 RNAseq data Experimental Models: Cell Lines2B4 cells retrovirally transduced Generated in the laboratory doi:with an NFAT-GFP reporter and of Dr. Marco Colonna. 10.1016/j.ce11.2015.retrovirally transduced with TREM2 01.049. 2B4 cells expressing NFAT-GFPGenerated in the laboratory doi: of Dr. Lewis Lanier10.1126/science.1070884 Experimental Models: Organisms/StrainsMouse: C57BL/6J WT The Jackson Laboratory Cat# 000664Mouse: 5XFAD/Tg6799 The Jackson Laboratory Cat# 34840- JAXMouse: Trem2^(-/-) Generated in the Laboratory of Dr. Marco ColonnaOligonucleotides For nuclear DNA FW: CTTCCCCACTGGCCTCAAG (SEQ ID NO: 3)RV: CCAAAACCCAGTGATCCAG C (SEQ ID NO: 4) For mitochondrial DNA FW:TGCTAGCCGCAGGCATTAC (SEQ ID NO: 5) RV: GGGTGCCCAAAGAATCAGAAC (SEQ ID NO: 6) For beta-Actin (RNAseq) FW: GGA GGG GGT TGA GGTGTT (SEQ ID NO: 7) RV: TGT GCA CTT TTA TTG GTC TCA AG (SEQ ID NO: 8)Linker primers for RNAseq CCTACACGACGCTCTT CCGATCT-XXXXXXXX-T15 (SEQ ID NO: 1) AGATCGGAAGAGCACA CGTCTG (SEQ ID NO: 2)Recombinant DNA Software and Algorithms Matlab MathWorks MorpheusBroad Institute Prism 7 Graphpad Fiji 2.0 ImageJ Imaris 7.7 Bitplane IPAQIAGEN Shiny GAM https://artyomovlab.wustl. edu/shiny/gam/ OtherXF96 Extracellular Flux Analyzer Agilent Nikon A1Rsi Confocal MicroscopeNikon

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All cited references are herein expressly incorporated by reference intheir entirety.

Whereas particular embodiments have been described above for purposes ofillustration, it will be appreciated by those skilled in the art thatnumerous variations of the details may be made without departing fromthe disclosure as described in the appended claims.

What is claimed is:
 1. A method for treating a microglialdysfunction-associated neurodegenerative disease in a subject in needthereof, the method comprising: administering to the subject atherapeutically effective amount of a composition comprising amicroglial rescuing agent, wherein the microglial rescuing agent is oneor more of a creatine, a creatine analog, a Dectin-1 agonist orpharmaceutically acceptable salt thereof.
 2. The method of claim 1,wherein the microglial dysfunction-associated neurodegenerative diseaseis Alzheimer's disease.
 3. The method of claim 1, wherein the microglialdysfunction-associated neurodegenerative disease is a neurodegenerativedisease characterized by SNPs or mutations effecting microglialfunction, a TREM2 variant, or an ApoE variant, resulting in decreasedmicroglial activity.
 4. The method of claim 3, wherein the microglialdysfunction-associated neurodegenerative disease is characterized bysingle nucleotide polymorphisms (SNPs) or mutation in Trem2 or ApoEaffecting microglial activity.
 5. The method of claim 1, wherein atherapeutically effective amount of a microglial rescuing agent resultsin one or more of improved microglial metabolic activity, decreasedmicroglial autophagy, reduced neurite dystrophy, decreased cell death,improved microglia viability or improved microglia numbers.
 6. Themethod of claim 2, wherein the therapeutically effective amount of amicroglial rescuing agent results in improved microglia clusteringaround Aβ plaques or reduced plaque-associated neurite dystrophy.
 7. Themethod of claim 1, wherein the microglial rescuing agent is one or moreof creatine, nicotinamide mononucleotide, cyclocreatine,phosphocyclocreatine, Zymosan, and Zymosan Depleted.
 8. The method ofclaim 1, wherein the subject is human.
 9. A method of reversing neuronaldamage in a subject having a microglial dysfunction-associatedneurodegenerative disease, wherein the microglial dysfunction-associatedneurodegenerative disease is characterized by a single nucleotidepolymorphisms (SNPs) or mutation in Trem2 or ApoE affecting microglialfunctions, the method comprising: administering to the subject atherapeutically effective amount of a composition comprising amicroglial rescuing agent, wherein the microglial rescuing agent is oneor more of a creatine, a creatine analog, a Dectin-1 agonist orpharmaceutically acceptable salt thereof.
 10. The method of claim 9,wherein a therapeutically effective amount of a microglial rescuingagent in one or more of improved microglial metabolic activity,decreased microglial autophagy, reduced neurite dystrophy, decreasedcell death, improved microglia viability or improved microglia numbers.11. The method of claim 9, wherein the microglial dysfunction-associatedneurodegenerative disease is Alzheimer's disease.
 12. The method ofclaim 11, wherein the therapeutically effective amount of a microglialrescuing agent results in improved microglia clustering around Aβplaques or reduced plaque-associated neurite dystrophy.
 13. The methodof claim 9, wherein the microglial rescuing agent is one or more ofcreatine, nicotinamide mononucleotide, cyclocreatine,phosphocyclocreatine, Zymosan, and Zymosan Depleted.
 14. The method ofclaim 9, wherein the subject has TREM2 deficient cells in the brainprior to administration of the microglial rescuing agent.
 15. The methodof claim 9, wherein the subject is human.
 16. A method of treating atleast one symptom of cognitive dysfunction in a subject having amicroglial dysfunction-associated neurodegenerative disease, wherein themicroglial dysfunction-associated neurodegenerative disease ischaracterized by a single nucleotide polymorphisms (SNPs) or mutation inTrem2 or ApoE affecting microglial functions, the method comprising:administering to the subject a therapeutically effective amount of acomposition comprising a microglial rescuing agent, wherein themicroglial rescuing agent is one or more of a creatine, a creatineanalog, a Dectin-1 agonist or pharmaceutically acceptable salt thereof.17. The method of claim 16, wherein at least one symptom comprises shortterm memory function.
 18. The method of claim 16, wherein the at leastone symptom comprises a spatial learning dysfunction.
 19. The method ofclaim 16, wherein the microglial rescuing agent is one or more ofcreatine, nicotinamide mononucleotide, cyclocreatine,phosphocyclocreatine, Zymosan, and Zymosan Depleted.
 20. The method ofclaim 16, wherein the subject is human.